U.S. patent number 10,551,371 [Application Number 12/774,709] was granted by the patent office on 2020-02-04 for system and method for monitoring cardiomyocyte beating, viability and morphology and for screening for pharmacological agents which may induce cardiotoxicity or modulate cardiomyocyte function.
This patent grant is currently assigned to ACEA Biosciences, Inc.. The grantee listed for this patent is Yama A. Abassi, Nan Li, Wei Ouyang, Xiaobo Wang, Biao Xi, Xiao Xu. Invention is credited to Yama A. Abassi, Nan Li, Wei Ouyang, Xiaobo Wang, Biao Xi, Xiao Xu.
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United States Patent |
10,551,371 |
Wang , et al. |
February 4, 2020 |
System and method for monitoring cardiomyocyte beating, viability
and morphology and for screening for pharmacological agents which
may induce cardiotoxicity or modulate cardiomyocyte function
Abstract
Devices and methods for performing extracellular recording of
cells, such as excitable cells, cardiomyocytes, and cardiomyocyte
precursor cells is provided. An exemplary device includes a
nonconductive substrate forming or provided as a base of one or
more wells; a recording electrode positioned on the substrate
within the well, wherein the recording electrode is accessible to
cells when a cell sample is added to the device; and a reference
electrode positioned within the well in a cell-free zone, the
cell-free zone characterized as free from contact with cells when
the cell sample is added to the device, thereby preventing contact
between cells and the reference electrode.
Inventors: |
Wang; Xiaobo (San Diego,
CA), Ouyang; Wei (San Diego, CA), Li; Nan (San Diego,
CA), Xi; Biao (San Diego, CA), Abassi; Yama A. (San
Diego, CA), Xu; Xiao (San Diego, CA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Wang; Xiaobo
Ouyang; Wei
Li; Nan
Xi; Biao
Abassi; Yama A.
Xu; Xiao |
San Diego
San Diego
San Diego
San Diego
San Diego
San Diego |
CA
CA
CA
CA
CA
CA |
US
US
US
US
US
US |
|
|
Assignee: |
ACEA Biosciences, Inc. (San
Diego, CA)
|
Family
ID: |
43588804 |
Appl.
No.: |
12/774,709 |
Filed: |
May 5, 2010 |
Prior Publication Data
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Document
Identifier |
Publication Date |
|
US 20110039294 A1 |
Feb 17, 2011 |
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Related U.S. Patent Documents
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Application
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Filing Date |
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Issue Date |
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12435569 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N
33/48735 (20130101) |
Current International
Class: |
G01N
33/487 (20060101) |
Field of
Search: |
;422/83 |
References Cited
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Apr 2017 |
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WO |
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2017/087945 |
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WO |
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Primary Examiner: Eom; Robert J
Attorney, Agent or Firm: Wagenknecht IP Law Group PC
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation in part of U.S. patent
application Ser. No. 12/435,569, filed May 5, 2009, entitled,
"Label free monitoring of excitation-contraction coupling and
excitable cells using impedance based systems with millisecond time
resolution," which claims priority to U.S. provisional patent
application Ser. No. 61/191,684, filed Sep. 11, 2008, and U.S.
provisional patent application Ser. No. 61/126,533, filed May 5,
2008; the contents of each are herein incorporated by reference in
their entirety.
This application also claims priority to U.S. patent application
Ser. No. 61/323,782, filed on Apr. 13, 2010, entitled, "Impedance
based monitoring of cardiomyocytes"; U.S. patent application Ser.
No. 61/310,557, filed Mar. 15, 2010, entitled, "Impedance based
monitoring of cardiomyocytes"; and U.S. patent application Ser. No.
61/175,566, filed May 5, 2009, entitled, "System and method for
monitoring cardiomyocyte beating, viability and morphology and for
screening for pharmacological agents which may induce
cardiotoxicity or modulate cardiomyocyte function." The contents of
each are herein incorporated by reference in their entirety.
This application is also a continuation in part of U.S. patent
application Ser. No. 11/235,938 entitled, "Dynamic Monitoring of
Cell Adhesion and Spreading Using the RT-CES System", filed on Sep.
27, 2005 which is a continuation in part of U.S. patent application
Ser. No. 11/197,994, now U.S. Pat. No. 7,468,255, entitled, "Method
for Assaying for Natural Killer, Cytotoxic T-Lymphocyte and
Neutrophil-Mediated Killing of Target Cells Using Real-Time
Microelectronic Cell Sensing Technology", filed on Aug. 4, 2005,
which is a continuation-in-part of U.S. patent application Ser. No.
11/055,639, now U.S. Pat. No. 7,560,269, entitled "Real time
electronic cell sensing system and applications for cytotoxicity
profiling and compound assays" filed on Feb. 9, 2005 which is a
continuation-in-part of U.S. patent application Ser. No.
10/987,732, now U.S. Pat. No. 7,192,752, entitled "Real time
electronic cell sensing system and application for cell based
assays" filed on Nov. 12, 2004, which claims priority to U.S.
provisional application Ser. No. 60/519,567, filed on Nov. 12,
2003.
All applications referred to in this paragraph are incorporated by
reference in their entireties herein.
Parent U.S. patent application Ser. No. 11/235,938, also claims
benefit of priority to U.S. provisional patent application Ser. No.
60/630,131, filed on Nov. 22, 2004; U.S. provisional patent
application Ser. No. 60/630,071 filed on Nov. 22, 2004; U.S.
provisional patent application Ser. No. 60/613,872 filed on Sep.
27, 2004; U.S. provisional patent application Ser. No. 60/613,749,
filed on Sep. 27, 2004; U.S. provisional patent application Ser.
No. 60/630,809 filed on Nov. 24, 2004; U.S. provisional patent
application Ser. No. 60/633,019 filed on Dec. 3, 2004; U.S.
provisional patent application Ser. No. 60/647,159 filed on Jan.
26, 2005; U.S. provisional patent application Ser. No. 60/653,904
filed on Feb. 17, 2005; and U.S. provisional patent application
Ser. No. 60/673,678 filed on Apr. 25, 2005; U.S. provisional patent
application Ser. No. 60/689,422 filed on Jun. 10, 2005. The
contents of each are herein incorporated by reference in their
entirety.
Parent U.S. patent application Ser. No. 11/235,938 is also a
continuation-in-part of U.S. patent application Ser. No.
11/198,831, entitled, "Dynamic Monitoring of Activation of
G-Protein Coupled Receptor (GPCR) and Receptor Tyrosine Kinase
(RTK) in Living Cells using Real-Time Microelectronic Cell Sensing
Technology, filed on Aug. 4, 2005, which is herein incorporated by
reference in its entirety.
Parent U.S. patent application Ser. No. 10/987,732, now U.S. Pat.
No. 7,192,752, is also a continuation-in-part of U.S. patent
application Ser. No. 10/705,615, now U.S. Pat. No. 7,459,303,
entitled "Impedance Based Apparatuses and Methods for Analyzing
Cells and Particles", filed on Nov. 10, 2003, which claims priority
to U.S. provisional patent application Ser. No. 60/397,749 filed on
Jul. 20, 2002; U.S. provisional patent application Ser. 60/435,400,
filed on Dec. 20, 2002; U.S. provisional patent application Ser.
No. 60/469,572, filed on May 9, 2003; and PCT patent application
serial number PCT/US03/22537, filed on Jul. 18, 2003. All of the
applications referred to in this paragraph are incorporated by
reference in their entireties herein.
Parent U.S. patent application Ser. No. 10/987,732, now U.S. Pat.
No. 7,192,752, is also a continuation in part of U.S. patent
application Ser. No. 10/705,447, now U.S. Pat. No. 7,470,533, filed
on Nov. 10, 2003, entitled "Impedance Based Devices and Methods for
Use in Assays" which claims priority to U.S. provisional patent
application Ser. No. 60/397,749, filed on Jul. 20, 2002; U.S.
provisional patent application Ser. No. 60/435,400, filed on Dec.
20, 2002; U.S. provisional patent application Ser. No. 60/469,572,
filed on May 9, 2003; and PCT patent application serial number
PCT/US03/22557, filed on Jul. 18, 2003. All of the applications
referred to in this paragraph are incorporated by reference in
their entireties herein.
Parent U.S. patent application Ser. No. 11/055,639, now U.S. Pat.
No. 7,560,269 also claims priority to U.S. provisional patent
application Ser. No. 60/542,927 filed on Feb. 9, 2004; U.S.
provisional patent application Ser. No. 60/548,713, filed on Feb.
27, 2004, and U.S. provisional patent application Ser. No.
60/614,601, filed on Sep. 29, 2004. All of the applications
referred to in this paragraph are incorporated by reference in
their entireties herein.
Parent U.S. patent application Ser. No. 11/197,994, now U.S. Pat.
No. 7,468,255 also claims priority to U.S. provisional patent
application Ser. No. 60/598,608, filed on Aug. 4, 2004, U.S.
provisional patent application Ser. No. 60/630,131, filed on Nov.
22, 2004, U.S. provisional patent application Ser. No. 60/689,422,
filed on Jun. 10, 2005, U.S. provisional patent application Ser.
No. 60/598,609, filed on Aug. 4, 2004, U.S. provisional patent
application Ser. No. 60/613,749, filed on Sep. 27, 2004, U.S.
provisional patent application Ser. No. 60/647,189, filed on Jan.
26, 2005, U.S. provisional patent application Ser. No. 60/647,075,
filed on Jan. 26, 2005, U.S. provisional patent application Ser.
No. 60/660,829, filed on Mar. 10, 2005, and U.S. provisional patent
application Ser. No. 60/660,898, filed on Mar. 10, 2005. All of the
applications referred to in this paragraph are incorporated by
reference in their entireties herein.
Claims
What is claimed is:
1. A system for parallel impedance monitoring and extracellular
field potential recording of cells, the system comprising: a) a
device comprising: i) a nonconductive substrate forming a base of
one or more wells, ii) a set of at least two impedance electrodes
capable of monitoring cell-substrate impedance of cells, the at
least two impedance electrodes positioned on the nonconductive
substrate within the one or more wells configured for
cell-substrate impedance monitoring at 10 millisecond resolution,
iii) a set of extracellular recording electrodes positioned on the
substrate within the one or more wells and configured for
extracellular field recording of the cells, wherein the set of at
least two impedance electrodes and the set of extracellular
recording electrodes are different sets of electrodes, further
wherein all of the electrodes are on a same surface; b) an
impedance analyzer for measuring cell-substrate impedance at 10
millisecond resolution; c) an extracellular field potential
amplifier for extracellular field potential recording; and d) a
switch that switches the one or more wells between measuring
impedance and extracellular field potential recording modes.
2. The system according to claim 1, wherein the at least two
impedance electrodes are interdigitated electrodes.
3. The system according to claim 2, wherein each of the at least
two impedance electrodes comprises an electrode structure
comprising a plurality of electrode elements, wherein the electrode
elements of different electrode structure are interdigitated.
4. The system according to claim 2, wherein the at least two
impedance electrodes and set of extracellular recording electrodes
share a same electrode.
5. The system according to claim 1, wherein the at least two
impedance electrodes and set of extracellular recording electrodes
share a same electrode.
6. The system according to claim 1, wherein impedance may be
monitored for cells positioned at either of the at least two
impedance electrodes, further wherein one of the at least two
impedance electrodes is configured as a reference electrode in the
set of extracellular recording electrodes.
7. A method of extracellular field potential recording and
impedance monitoring of a cell population, the method comprising:
a) providing the system according to claim 1; b) adding a cell
sample to the device; c) performing extracellular field potential
recording measurements of the cell sample by amplifying the voltage
signals between the set of extracellular recording electrodes and
monitoring the amplified signals; and d) performing impedance
measurements by monitoring cell-substrate impedance between the two
impedance electrodes at 10 millisecond resolution.
8. The method according to claim 7, wherein the cells comprise
cardiomyocytes or cardiomyocyte precursor cells, the method further
comprising adding a compound suspected of affecting a beating cycle
of the cells.
9. The method according to claim 8, further comprising resolving
polarization and depolarization of beating cells.
10. The method according to claim 8, further comprising determining
whether or not the compound affects cell beating.
11. The method according to claim 9, further comprising resolving a
beating cycle of the cells from the impedance measurements.
12. The method according to claim 9, further comprising determining
a beating rate from the impedance measurements.
13. The method according to claim 9, further comprising determining
a beating amplitude from the impedance measurements.
14. The method according to claim 9, wherein the step of performing
impedance measurements is performed at 5 millisecond
resolution.
15. The method according to claim 9, wherein two consecutive
impedance measurements are performed at 1 millisecond or
faster.
16. The method according to claim 7, further comprising calculating
a cell index parameter from measured impedance values.
17. The method according to claim 7, wherein the cells comprise
cardiomyocytes or cardiomyocyte precursor cells, the method further
comprising resolving a beating cycle of the cells from the
impedance measurements.
18. The method according to claim 7, wherein the cells comprise
cardiomyocytes or cardiomyocyte precursor cells, the method further
comprising determining a beating rate from the impedance
measurements.
19. The method according to claim 7, wherein the cells comprise
cardiomyocytes or cardiomyocyte precursor cells, the method further
comprising determining a beating amplitude from the impedance
measurements.
20. The method according to claim 7, wherein the step of performing
impedance measurements is performed at 5 millisecond
resolution.
21. The method according to claim 7, wherein two consecutive
impedance measurements are performed at 1 millisecond or faster.
Description
TECHNICAL FIELD
This invention relates to the field of cell-based assays and more
specifically to devices, methods and systems for performing
extracellular recording of cells, such as cardiomyocytes, alone or
in parallel with impedance monitoring.
BACKGROUND OF THE INVENTION
A. Electronic Analysis of Cells
Bioelectronics is a progressing interdisciplinary research field
that involves the integration of biomaterials with electronic
devices. Bioelectronics methods have been used for analyzing cells
and assaying biological molecules and cells. In one type of
application, cells are cultured on microelectrodes and
cell-electrode impedance is measured and determined to monitor
cellular changes.
In PCT Application No. PCT/US03/22557, titled "IMPEDANCE BASED
DEVICES AND METHODS FOR USE IN ASSAYS", filed on Jul. 18, 2003, a
device for detecting cells and/or molecules on an electrode surface
is disclosed. The device detects cells and/or molecules through
measurement of impedance changes resulting from the attachment or
binding of cells and/or molecules to the electrode surfaces. A
number of embodiments of the device are disclosed, together with
the apparatuses, and systems for using such devices to perform
certain cell based assays.
B. Dynamic Monitoring of Cardiomyocyte Viability, Morphology and
Beating In Vitro
To bring a new drug to the market it can take anywhere between 8 to
16 years, and average cost of developing a drug is now around $500
m-$800 m with the cost expected to hit the $1 bn mark within the
next four years. Cardiotoxicity has been cited as the reason for 30
percent of all failed drug compounds during development and is a
major cause of compound attrition. The late detection of
cardiotoxic side effects caused by pharmacological compounds can
impede drug discovery and development projects, and consequently
increase their cost. Testing for the potential cardiotoxic side
effects of compounds at an early stage of drug development has
therefore been the goal of many pharmaceutical and biotechnology
companies. Cardiotoxicity itself can entail a number of short-term
and long term cellular events including directly affecting the
beating rate of cardiomyocytes, viability of cardiomyocytes and
morphology of cardiomyocytes as would occur in hypertrophy. The
core of the current issue in pharmacological safety assessment and
drug development is the lack of a reliable screening methodology
capable of monitoring potential drug-mediated cardiotoxicity and
distinguishing between different modes of cardiotoxicity. What is
urgently needed in the field is a good cell-based model system as
well as a monitoring system with a physiological and functional
readout that can provide incisive information regarding potential
cardiotoxic side effects of drugs.
1. Cell-Based Model System for Studying Cardiac Function
Traditionally, the drug discovery industry has undertaken two
different approaches for toxicological assessment of drug candidate
leads in cardiac function. The first approach involves isolation of
cardiomyocytes directly from a mammalian species such as rats and
dogs followed by electrophysiological and viability studies on the
isolated cardiomyocytes. This approach is extremely
labor-intensive, time consuming and costly and at the same time not
very amenable to the high throughput demands of pharmaceutical
industry.
An alternative method for prediction of cardiotoxicity of drug
candidate leads early in the drug development process has involved
utilizing cell-based assay models which heterologously express
specific ion channels such as hERG channels or voltage-gated
calcium channels. These cardiac ion channels have been envisioned
as possible molecular targets through which drugs could induce
cardiotoxicity. These cell-based systems allow the assessment of
drug-channel interaction by monitoring the effect of the drug on
the currents produced by the different channels in cultured cells
using a technique known as `patch clamping`, which isolates regions
of the cell membrane containing channel proteins and measures
changes in electrical potential difference. Use of this method in
high throughput requires automation of patch clamping in array
format, which even though is available in last several years but is
not yet widespread. The other issue with this approach is that
cardiac toxicity may occur by other mechanisms which can easily be
missed by this type of targeted approach. An alternative to the in
vitro ion-channel recording assays as well as the labor-intensive
isolation of primary tissue is the differentiation of embryonic
stem (ES) cells into cardiomyocytes as a starting material for
functional assays.
The utility of ES cells as a treatment for various chronic diseases
has received much attention in recent years. Mammalian ES cells are
self renewing cells derived from the inner cell mass of a
blastocyst stage embryo which can be differentiated into multiple
different cell types. It has been demonstrated that the mouse ES
cells as well as human ES cells can be differentiated into
cardiomyocytes which retain the ability to beat in culture. The
differentiation of ES cells first involves an intermediate in vitro
developmental stage in which ES cells form compact cell structures
known as embryoid bodies. These embryoid bodies can induce the
developmental program of ES cell differentiation into multiple cell
types including cardiomyocytes which are distinguished in culture
by their ability to undergo spontaneous beating. These ES derived
in vitro differentiated cardiomyocytes recapitulates the normal
development of cardiomyocytes as evidenced by the stage-specific
expression of cardiomyocyte specific genes. All the known
transcription factors, ion channels and structural proteins that
are part of normal heart development and function in vivo are also
expressed in ES-derived cardiomyocyte.
Because ES cells are self renewing cells in culture they can serve
as an excellent source for continuous production of cardiomyocytes.
Therefore, these cardiomyocytes which behave in every way like
normal cardiomyocytes isolated from the heart tissue itself
addresses the ever important supply problem and for the first time
allows for assessment of cardiac function and its modulation by
lead candidate drugs and compounds in relatively large scale in
both viability assays, assessment of morphology and in monitoring
the beating function of cardiomyocytes. Furthermore, because the
technology exists to selectively knockout or express trans-genes in
ES cells, it provides an excellent model system to study the role
of certain genes in cardiac development and function without having
to be concerned about adverse affects on overall embryonic
development in transgenic animals. The ability to express
transgenes in ES cells has been utilized as a way to enrich for
preparation of cardiomyocytes that are 100% pure. For example, the
gene encoding GFP has been cloned downstream of a cardiac-specific
promoter and then introduced into ES cells. Embryoid cells which
ultimately differentiate into cardiomyocytes will therefore express
the GFP transgenes and these cells can be easily isolated by cell
sorting techniques and therefore an enriched cardiomyocyte
population can be obtained.
2. Technologies Used for Assessment of Cardiomyocyte Function In
Vitro
Technologies designed to assess cardiomyocyte behavior and function
and the effect of drugs and other manipulations in vitro can be
divided into two different approaches. One approach involves long
term assessment of cardiomyocyte viability for example in response
to certain compounds. Such assays are typically end point assays
designed to measure a cellular component such as ATP which
correlates with the degree of viability of the cells. The other
approach involves studying short term effect of drugs and compounds
on beating function of cardiomyocytes. High throughput techniques
for short term functional characterization of ion channels and
other targets in cardiomyocytes has been rather challenging and
limited. The systems such as automatic patch clamp instrumentation
that are available can monitor a single cardiomyocyte at a time and
with very limited throughput.
Accordingly, while there exists different approaches to assess
cardiotoxicity of various compounds, each suffers from one or more
drawbacks. Thus, there remains a need to develop improved devices
and methods that have high throughput capabilities and improved
accuracy and reliability with respect to measurements and
correlation with cell phenotype.
SUMMARY OF THE INVENTION
The present invention discloses devices, systems and methods for
performing extracellular recording of excitable cells, such as
cardiomyocytes, in vitro. Specifically, the devices and methods
provide improved characterization of excitable cells in response to
compound administration to assess cardiotoxic effects, while
increasing efficiency of throughput for potential drug candidates.
The invention further provides improved characterization of cells
during differentiation processes related to development of
cardiomyocytes.
It is an object of the invention to provide extracellular recording
configurations, which permit reproducible sampling across larger
cell populations compared to traditional approaches. It is another
object of the invention to provide cell-free zones for positioning
a reference electrode to prevent its direct interaction with a cell
population loaded onto the device. It is another object of the
invention to provide a combined device capable of both performing
extracellular recording measurements and impedance monitoring,
which may be selectively activated for measurement and thus provide
a multi-faceted system for assessing or characterizing excitable
cells.
In one aspect of the present invention a device for performing
extracellular recording of cells, such as excitable cells,
cardiomyocytes, and cardiomyocyte precursor cells is provided. An
exemplary device includes a) a nonconductive substrate forming or
provided as a base of one or more wells; b) a recording electrode
positioned on the substrate within the well, wherein the recording
electrode is accessible to cells when a cell sample is added to the
device; and c) a reference electrode positioned within the well in
a cell-free zone, the cell-free zone characterized as free from
contact with cells when the cell sample is added to the device,
thereby preventing contact between cells and the reference
electrode. The cell-free zone prevents direct contact between cells
from a cell sample and the reference electrode. In some
embodiments, the recording electrode has a diameter from about 10
um to about 200 um. In other embodiments, the recording electrode
comprises an electrode structure comprising a plurality of
electrode elements
In some embodiments, the cell-free zone is physically defined by
the presence of a barrier, such as a wall, through which cells do
not pass. In certain embodiments, the barrier could be in the form
of a removable blockage material which is placed directed on the
cell-free zone when cells are added to the well and which can be
removed after cells have settled along the bottom of the well.
Thus, the blocking structure may be in part removable. In other
embodiments, the cell free zone is provided as a platform or
surface positioned above the base substrate. In other embodiments,
the cell free zone is positioned along an angled wall, which is
angled upward from the bottom of the well. In other embodiments,
the cell-free zone is positioned above the substrate and within the
volume of the well such that an external electrode extends downward
into sample media, optionally from a lid or cap for the well.
In some embodiments the extracellular recording electrode is a
unitary or unbranched electrode and may be of a simple geometry
such as a circle, a square and the like. In other embodiments, the
extracellular recording electrode has a branched configuration,
which may permit extracellular recording over a larger cell
population. Increasing the measured cell population for
extracellular recording may increase measurement accuracy or
reproducibility of the recorded signals. In one exemplary
embodiment, a device for performing extracellular recordings of
excitable cells in vitro includes a) a nonconductive substrate; b)
one or more wells on the substrate; c) one or more electrode
structures fabricated on the substrate, each electrode structure
associated with one of the one or more wells; and d) one or more
reference electrodes external to the substrate, each of which can
be inserted into one of the one or more wells, wherein for each of
the one or wells, the corresponding electrode structures and
reference electrodes form an electrode pair. In further embodiments
the substrate has a surface suitable for attachment of excitable
cells, wherein the attachment of excitable cells on the substrate
can result in a detectable extracellular recording potential
between each electrode pair. In a further embodiment, which is also
a preferred embodiment, each electrode structure comprises multiple
electrode elements. In another preferred embodiment, each electrode
structure comprises multiple electrode elements, forming half of an
interdigitated electrode array. In another preferred embodiment,
each electrode structure comprises multiple electrode elements
which have different electrode element shapes, including
rectangular shape, or sinusoidal shape, or circle-on-line
shape.
In another preferred embodiment of the device for performing
extracellular recording, each electrode structure occupies a
substantial percentage of surface area of the well that the
electrode structure is associated with. In some preferred
embodiments, percentage of surface area of the well at the bottom
being occupied by the electrode structures is more than 1%.
Preferably, percentage of surface area of the well at the bottom
being occupied by the electrode structures is more than 5%.
Preferably, percentage of surface area of the well at the bottom
being occupied by the electrode structures is more than 10%. More
preferably, the percentage of surface area of the well at the
bottom being occupied by the electrode structures is more than 20%.
More preferably, the percentage of surface area of the well at the
bottom being occupied by the electrode structures is more than 30%.
More preferably, the percentage of surface area of the well at the
bottom being occupied by the electrode structures is more than 50%.
More preferably, the percentage of surface area of the well at the
bottom being occupied by the electrode structures is more than 70%.
More preferably, the percentage of surface area of the well at the
bottom being occupied by the electrode structures is more than
85%.
In another aspect of the present invention, a method of performing
extracellular recording of excitable cells includes, providing a
device of the present invention, adding excitable cells to the
wells; providing an extra-cellular-recording amplifier that can
measure and record voltage signals at microvolt levels, connecting
electrodes on the devices to the extra-cellular-recording
amplifier; monitoring and recording extracellular potentials at the
electrodes of the devices. Preferably, the method further comprises
analyzing recorded extracellular potential waveforms. More
preferably, the method further comprises adding a compound to the
well, measuring and recording extracellular potentials prior to and
after the compound addition; and analyzing extracellular potential
waveforms.
In still another aspect of the present invention, a device allowing
for parallel extracellular recording and cell impedance monitoring
of a cell population is provided. The device includes a cell
impedance monitoring means and an extracellular recording means
operably coupled through a same substrate. In a further embodiment
a switching means switches operation of the device between
impedance monitoring and extracellular recording.
In still another aspect of the present invention, a device allowing
for parallel extracellular recording and cell impedance measurement
comprises a nonconductive substrate, having a surface suitable for
attachment of excitable cells, one or more wells on the substrate;
for each well, a pair of impedance measurement electrodes, wherein
the attachment of excitable cells on the substrate can result in a
detectable impedance change between the pair of impedance
measurement electrodes; for each well, a pair of extra-cellular
recording electrodes, wherein the attachment of excitable cells on
the substrate can result in a detectable extra-cellular recording
potentials between the pair of extra-cellular recording electrodes.
Preferably, the pair of impedance measurement electrodes is located
on the substrate. More preferably, for the pair of extra-cellular
recording electrodes, one recording electrode is located on the
substrate and another electrode is located external to the
substrate. In other preferred embodiments, the reference electrode
may be isolated from cells by positioning the reference electrode
at a cell-free zone, such that it is a cell-free electrode. The
reference electrode may or may not be located on the substrate. The
device may also include or be operably connected to a switching
means to switch from an impedance mode to an extracellular
recording mode.
In another aspect of the invention, a device allowing for parallel
extracellular recording and cell impedance measurement includes a
nonconductive substrate forming or provided as a base of one or
more wells; at least two impedance electrodes capable of monitoring
impedance of the cells, the at least two impedance electrodes
positioned within a well and on the nonconductive substrate; and a
reference electrode, wherein a first impedance electrode from the
at least two impedance electrodes is electrically coupled to the
reference electrode for performing an extracellular recording
measurement at the first impedance electrode.
In still another aspect of the present invention, a method for
parallel measurement of cell-substrate impedance and extra-cellular
potentials includes a) providing a device allowing for parallel
extracellular recording and cell impedance measurement; b) adding
excitable cells to the wells of the device; c) providing an
impedance analyzer; d) providing an extracellular potential
amplifier; e) connecting impedance-measurement electrodes of the
devices to the impedance analyzer; f) connecting extra-cellular
recording electrodes of the devices to the extra-cellular potential
amplifier; g) performing parallel measurement of cell-substrate
impedance and extracellular potentials. Preferably, the method
further comprises adding compounds to the cells and monitoring
cell-substrate impedance and extracellular potentials prior to and
after compound addition. Preferably, the method further comprises
analyzing measured time dependent impedance responses. Still
preferably, the method further comprising analyzing
extracellular-recording wave forms.
In present invention, we describe label-free methods for monitoring
cardiomyocytes in vitro. In one aspect of the present invention,
extracellular-recording measurement, or parallel impedance and
extracellular recording measurement of cardiomyocytes on
microelectrodes is used to non-invasively monitor cardiac cell
viability in vitro in long term experiments. This method will allow
the continuous monitoring of cardiomyocyte viability overtime and
can monitor the interaction of compounds which ultimately result in
promoting loss of cardiomyocyte viability.
In another aspect of the present invention, extracellular-recording
measurement or parallel impedance and extracellular recording
measurement of cardiomyocytes on microelectrodes is used to
non-invasively monitor cardiomyocyte beating in vitro. The system
is capable of continuously monitoring the excitation-contraction
coupling, otherwise know as beating, of cardiomyocytes in a
relatively high-throughput manner using either
extracellular-recording measurement or parallel impedance and
extracellular recording measurement. It can be used for
pharmacological safety assessment, screening for novel compounds
which modulate cardiomyocyte function in a specific manner and
assessment of genes involved in cardiac function.
In another aspect of the present invention, extracellular recording
measurement or parallel impedance and extracellular recording
measurement can be used to monitor both short term beating whilst
impedance measurement can be used to monitor long term viability
status of cardiomyocytes in the same well. The ability to monitor
both cardiomyocyte beating in conjunction with viability for the
same population of cardiomyocytes over time would be an added
advantage. Certain manipulations such as drug treatment may not
manifest their effect on cardiomyocyte beating and/or viability
until at a later time period. Therefore, the time resolution as
well as the capabilities of extracellular recording measurements
together with impedance measurements of the system of the present
invention can be used to monitor cardiomyocyte beating and
viability over time.
In another aspect of the present invention, impedance readout can
be used to monitor the morphological or differentiative behavior of
cardiomyocytes in vitro. Certain treatments can induce changes in
morphological behavior of cardiomyocytes, such as inducing
hypertrophy which is associated with cardiomyocyte elongation and
expansion. Because impedance monitoring can detect changes in cell
morphology, it can be used to for detection of hypertrophy in
cardiomyocytes.
In another aspect of the present invention, direct optical
monitoring of cardiomyocytes is used to quantify and measure the
beating of cardiomyocytes. Still, in another aspect of the present
invention, extracellular recordings (i.e. measurement of electrical
potentials of extracellular recording electrodes when
cardiomyocytes are attached to the electrode surfaces) are used to
quantify and measure the beating of cardiomyocytes. Still, in
another aspect of the present invention, a physical method which
can be used to monitor cell-substrate interaction is used to
quantify and monitor beating of cardiomyocytes.
In another aspect of the present invention, the methods include
real-time impedance monitoring and/or extracellular recording of
cardiomyocyte in vitro. This invention will be able to monitor
viability of cardiomyocytes, the field potential of cardiomyocytes,
or the excitation-contraction coupling or beating and morphological
and differentiative aspects of cardiomyocytes in a label-free
manner and real-time manner. Furthermore, the invention will be
able to monitor cardiotoxicity and the effect of compounds and
drugs for safety pharmacology purposes and can also serve as a
screen for agents which can modulate cardiomyocyte function. This
system can be used in conjunction with ES derived cardiomyocytes,
adult stem-cell derived cardiomyocytes as well as primary
cardiomyocytes to study the role of different genes and proteins in
cardiac function and development.
The system of the present invention having the capability of
parallel impedance-based monitoring as well as extra-cellular
recording based monitoring of cardiomyocytes fills a major
technological gap in monitoring cardiomyocytes in vitro. At present
to our knowledge there are only a few technologies that can monitor
cardiomyocyte population function in vitro, especially with regards
to cardiotoxicity and are limited in their throughput. In addition
to functional monitoring of cardiomyocyte beating the current
invention offers several other major benefits which are worth
discussing. Among these include the impedance system described here
can monitor cardiomyocytes for short durations, milliseconds to
minutes and long durations, several hours to days. Therefore, both
short term and long term effects of drugs not only on cardiomyocyte
beating, but viability and changes in morphology and adhesion can
also be assessed. This feature is especially important because
certain compounds such as .beta.-2 adrenergic receptor agonists,
well known and characterized modulators of heart function in vivo
and in vitro, can induce long term hypertrophic responses in
cardiomyocytes, which is associated with elongated morphology of
the cells. In addition, the systems of the present invention can
also record cardiomyocyte action potentials or field potentials,
allowing detailed electrophysiological analysis and assessment of
cardiomyocytes for relatively short duration at a time.
The method of the present invention is to devise a label-free
cell-based assay system for continuous monitoring of cardiomyocyte
viability, the rhythmic beating of cardiomyocytes and cardiomyocyte
morphology and differentiation and to screen for pharmacological
agents which may modulate these processes and induce
cardiotoxicity. In one aspect, viability is monitored based on long
term impedance monitoring of cardiomyocytes seeded in
microelectronic plates (E-PLATES), and in parallel, the field
potential generated by the excitation of the cardiomyocytes or
other excitable cells is monitored using the extracellular
potential recording electrodes associated with the wells of the
E-PLATES. Viable cells will continue to generate impedance signal
and any changes in viability, especially due to cytotoxic or
cardiotoxic drugs will be reflected by changes in impedance. In one
respect, the method includes providing a device for measuring
cell-substrate impedance and extracellular potentials operably
connected to an impedance analyzer and extracellular recording
potential amplifier, wherein the device includes at least one well
optionally coated with fibronectin or other appropriate
extracellular matrix proteins (e.g. gelatin) to expedite
attachment; adding cells to the at least one well, where the cells
can be mouse or human or other mammalian ES cells or adult stem
cells destined to differentiate into cardiomyocytes or primary
cardiomyocytes isolated directly from the heart of an experimental
system including mice, rats, rabbits or dog; monitoring impedance
of the at least one well over a period of time and optionally
determining cell index from impedance values; and monitoring the
extracellular potentials using recording electrodes associated with
the wells of the E-PLATES.
In another aspect, the method includes providing a device for
measuring cell-substrate impedance and for performing extracellular
recording operably connected to an impedance analyzer and
extracellular recording potential amplifier, wherein the device
includes at least two wells optionally coated with fibronectin (or
other extracellular matrix proteins such as gelatin) to expedite
attachment; adding cells to the at least two wells, where the cells
can be mouse or human or other mammalian ES cells or adult stem
cells destined to differentiate into cardiomyocytes or primary
cardiomyocytes isolated directly from the heart of an experimental
system including mice, rats, rabbits or dog; monitoring impedance
and monitoring extracellular potentials of the at least two wells
over a period of time. Treating at least one well with an agent;
where the agent could include but is not limited to a compound,
peptide, protein, antibody, siRNA, shRNA, lipid or any combination
of thereof and the other well is treated with an appropriate
control; continue monitoring of control and treated well over a
period of time preoptimized for the experiment of interest;
concluding that the factor may affect cell viability if the
impedance of the treated well is significantly different than the
impedance of the treated well and/or concluding that the factor may
affect cell excitation if the extracellular potential of the
treated well is significantly different from that of the treated
well.
Dynamic monitoring of cardiomyocyte beating is based on
quantification in real time of the rhythmic changes in
cardiomyocyte morphology as a result of the excitation contraction
coupling of the electrically excitable cardiomyocytes growing on
microelectrodes' surface in ACEA E-PLATES. The quantification of
the rhythmic changes in cardiomyocyte morphology is achieved via
the fast (down to milliseconds level, for example, at 40
milli-second, 30 milli-second, 20 milli-second, 10 millisecond or 5
milli-second time resolutions) and continuous measurement of
electrode impedance. The method essentially provides a cellular
cardio-gram which can provide incisive information about the status
of cardiomyocytes especially upon treatment with pharmacological
agents. In addition, the method allows for parallel measurement of
extracellular potentials of beating cardiomyocytes, providing
important electro-physiological property information of the cells.
The method includes providing a device for measuring cell-substrate
impedance and for monitoring extracellular potential operably
connected to an impedance analyzer and an extracellular potential
amplifier, wherein the device includes at least one well optionally
coated with fibronectin (or other suitable extracellular matrix
proteins) to expedite attachment; adding cells to the at least one
well, where the cells can be mouse or human or other mammalian ES
cells or adult stem cells destined to differentiate into
cardiomyocytes or primary cardiomyocytes isolated directly from the
heart of an experimental system including mice, rats, rabbits or
dog; monitoring impedance of the at least one well at time
intervals over a period of time and optionally determining cell
indices from impedance values and monitoring extracellular
potentials from the at least one well for a time period (for
example, 10 seconds) at certain time intervals (for example, every
10 minutes); optionally calculating average rate of beats per unit
time, average amplitude intensity in a unit time as well as the
average length of time between the beats.
In another aspect, the present invention is directed to method to
screen for potential agents that modulate ES-derived cardiomyocyte
beating, adult stem cell-derived cardiomyocyte beating or primary
cardiomyocyte beating by monitoring and measuring the
excitation-contraction coupling of cardiomyocyte and its modulation
by the agent. The agent may include but is not limited to
compounds, drugs, peptides, proteins, antibodies, siRNA, shRNA,
miRNA, cDNA, lipids and any combination thereof. The method
includes providing a device for measuring cell-substrate impedance
and for monitoring extracellular potentials operably connected to
an impedance analyzer and to an extracellular potential amplifier,
wherein the device includes at least two wells; adding ES cells,
adult stem-cell derived cardiomyocytes or primary cardiomyocytes to
at least two wells; monitoring impedance of the at least two wells
at different or similar time intervals over a period of time and
optionally determining cell indices from impedance values and
monitoring and recording extracellular potentials of the at least
two wells for a time window at time intervals over a period of
time; generating an impedance-based curve or optionally a cell
index curve for each of the at least one known factor and the
control; comparing the impedance-based curves or optionally the
cell index curves between the at least one known biologically
active agent well and the control well; the impedance-based curves
could be direct measurement of cardiomyocyte excitation-contraction
coupling and if significantly different, concluding that the
biologically active agent modulates cardiomyocyte function, and/or
comparing the extracellular potential waveforms from the active
agent well and the control well and, if significantly different,
concluding that the biologically active agent modulates the
excitation property of the cardiomyocytes. Optionally,
impedance-based curves or optionally cell index curves are used to
calculate average rate of beats of cardiomyocytes per unit time,
average amplitude intensity in a unit time as well as the average
length of time between the beats, comparison of these optionally
derived parameters is made between the at least one known
biologically active agent well and the control well, and if
significant differences exist, one may optionally conclude that the
biologically active agents modulate cardiomyocyte functions.
It is well established that certain pharmacological treatments and
disease conditions can result in cardiac hypertrophy or atrophy
culminating in changes in the morphology of cardiomyocyte. Cell
substrate impedance can be used to precisely measure and quantify
these changes in cell morphology and shape. Certain treatments can
also affect the differentiative process of ES cells to
cardiomyocytes which may involve specific morphological and
adhesive changes. In another aspect, the present invention is
directed to method to screen for potential agents that may modulate
the morphology of ES-derived cardiomyocyte, adult stem cell-derived
cardiomyocyte or primary cardiomyocyte or its differentiation. The
agent may include but is not limited to compounds, drugs, peptides,
proteins, antibodies, siRNA, shRNA, miRNA, cDNA, lipids and any
combination thereof. The method includes providing a device for
measuring cell-substrate impedance operably connected to an
impedance analyzer, wherein the device includes at least two wells;
adding ES cells, adult stem-cell derived cardiomyocytes or primary
cardiomyocytes to at least two wells; monitoring impedance of the
at least two wells at different or similar time intervals over a
period of time and optionally determining cell indices from
impedance values; generating an impedance-based curve or optionally
a cell index curve for each of the at least one known factor and
the control; comparing the impedance-based curves or optionally the
cell index curves between the at least one known biologically
active agent well and the control well; the impedance-based curves
could be direct measurement of changes in cell morphology and if
significantly different, concluding that the biologically active
agent modulates cardiomyocyte function. Preferably, the method
further comprises providing the device of the present invention
operably connected to an extracellular potential amplifier,
monitoring extracellular potentials of cells from one biologically
active agent well and one control well. Optionally, impedance-based
curves or optionally cell index curves are used to calculate the
compound dose-dependent changes in cardiomyocyte morphology and
generate an EC-50 value for the potency of the compound. In
addition, extracellular recording permits comparisons with cells at
various developmental stages to assess development.
In another aspect, the present invention is directed to method to
establish an assay to assess the effect of gene knockout or
transgene expression in ES cells differentiated to cardiomyocytes
and functionally monitored by the system of the present invention.
The method includes providing a device for measuring cell-substrate
impedance and for monitoring extracellular potential operably
connected to an impedance analyzer and extracellular potential
amplifier, wherein the device includes at least two wells; adding
wildtype ES cells as control to at least 1 well and ES cells with a
gene knockout or a transgene in at least 1 other well; monitoring
impedance of the at least two wells at time intervals over a period
of time and optionally determining cell indices from impedance
values; comparing the impedance-based curves or optionally the cell
index curves between the control well and the well containing the
ES cells harboring a kockout of a specific gene or expressing a
specific transgene; and if significantly different, concluding that
the gene knockout or the transgene can affect either cardiomyocyte
viability, morphology from ES cells or cardiomyocyte function as
monitored by observing the excitation-contraction coupling, and
alternatively, monitoring and recording extracellular potentials
from the at least two wells for a time period, comparing the
extracellular potential waveforms between the control well and the
well containing the ES cells harboring a kockout of a specific gene
or expressing a specific transgene; and if significantly different,
concluding that the gene knockout or the transgene can affect
cardiomyocyte electrophysiological property of the
cardiomyocytes.
In another aspect of the present invention, direct optical
monitoring of cardiomyocytes is used to quantify and measure the
beating of cardiomyocytes. The method includes providing a device
for optically monitoring cells and monitoring cell morphology
operably connected to an optical measurement system, where the
device includes at least two wells optionally coated with
fibronectin (or other suitable extracellular matrix proteins) to
expedite attachment; adding cells to the at least two wells, where
the cells can be mouse or human or other mammalian ES cells
destined to differentiate into cardiomyocytes or primary
cardiomyocytes isolated directly from the heart of an experimental
system including mice, rats, rabbits or dog; optically monitoring
the cells of at least two wells at time intervals over a period of
time via the optical measurement system; optionally calculating
average rate of beats per unit time, average amplitude intensity in
a unit time as well as the average length of time between the
beats. The device for such optical measurement of cells may include
microtiter plates. The optical system may include optical
magnification instrument such as microscope, optical CCD camera,
optical-signal processing algorithm to quantify cell beating and to
derive cell-beating parameters (such as calculating average rate of
beats per unit time, average amplitude intensity in a unit time as
well as the average length of time between the beats) based on cell
morphology images.
In another aspect of the present invention, extracellular
recordings (i.e. measurement of electrical potentials of
extracellular recording electrodes when cardiomyocytes are attached
to the electrode surfaces) are used to quantify and measure the
beating of cardiomyocytes. The method includes providing a device
with microelectrodes for extracellular recording of multiple cells
operably connected to an extracellular potential recording system,
where the device includes at least two wells optionally coated with
fibronectin (or other extracellular matrix proteins) to expedite
attachment; adding cells to the at least two wells, where the cells
can be mouse or human or other mammalian ES cells destined to
differentiate into cardiomyocytes or primary cardiomyocytes
isolated directly from the heart of an experimental system
including mice, rats, rabbits or dog; performing extracellular
recording of the cells of at least two wells via the extracellular
recording system; optionally calculating average rate of beats per
unit time, average amplitude intensity in a unit time as well as
the average length of time between the beats. The device for such
extracellular recording system includes microelectrode arrays or
structures inside wells of microtiter plates. The microelectrode
array may comprise two electrode structures having the
substantially same surface area and may be located along the
majority of the surface of the wells. The extracellular recording
system may include electronic measurement circuits capable of
recording small extracellular potentials induced on
microelectrodes, where cells are cultured. The signals from the
extracellular recording can be used to derive the average rate of
beats of cardiomyocytes per unit time, average amplitude intensity
in a unit time as well as the average length of time between the
beats, or other electrophysiologically-associated parameters.
Still, in another aspect of the present invention, a physical
method which can be used to monitor cell-substrate interaction is
used to quantify and monitor beating of cardiomyocytes. The method
includes providing a device for physically monitoring cells and
monitoring cell morphology operably connected to a physical
measurement system, where the device includes at least two wells
optionally coated with fibronectin (or other extracellular matrix
proteins) to expedite attachment; adding cells to the at least two
wells, where the cells can be mouse or human or other mammalian ES
cells destined to differentiate into cardiomyocytes or primary
cardiomyocytes isolated directly from the heart of an experimental
system including mice, rats, rabbits or dog; optically monitoring
the cells of at least two wells at time intervals over a period of
time via the physical measurement system; optionally calculating
average rate of beats per unit time, average amplitude intensity in
a unit time as well as the average length of time between the
beats. The device for such physical measurement of cells shall have
such property that the interaction between cells and substrate
surfaces can be monitored. Depending which physical method is used
for monitoring cell-substrate interaction, different devices may be
used. For example, the device can be an optical sensor, which can
be used to detect and measure biological reactions. One example of
optical sensor is resonant waveguide, which consists of a substrate
with an optical grating and a coating with a high refraction index.
This grating forms an optical waveguide, a tiny channel through
which a specific wavelength of light propagates. Only this specific
wavelength that is resonant with the waveguide grating structure is
strongly reflected. In this case, the physical measurement system
can detect changes in refractive index upon cell-substrate
interaction events or upon intracellular events. Examples of
cell-substrate interaction events or intracellular events may
include those taking place in cultured cells stimulated by a
compound treatment.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the cell index curves measured on RT-CES system for 4
different seeding densities (3750, 7500, 15,000 and 30,000 cells
per well) of mouse ES-derived cardiomyocytes. Cell index is a
dimensionless parameter derived in RT-CES system.
FIG. 2A shows the time dependent, normalized cell index for
embryonic stem cell derived cardiomyocytes in different wells
treated with different concentrations of sodium dichromate
dehydrate. FIG. 2B shows the dose dependent normalized cell index
values at 24 hrs after treatment of the cells with dichromate
dehydrate as a function of the concentration of dichromate
dehydrate.
FIG. 3A shows that the time dependent, normalized cell index for
embryonic stem cell derived cardiomyocytes in different wells
treated with different concentrations of isoproteranol. FIG. 3B
shows the dose dependent, normalized cell index at several hours
after treatment of the cells with isoproteranol as a function of
the concentration of isoproteranol.
FIG. 4 shows the cell index curves of ES-derived cardiomyocytes
measured on RT-CES system for 4 individual wells, with cell culture
medium as background starting from cell seeding to about 86 hrs
after cell seeding.
FIG. 5 shows the detection of cardiomyocyte beating using the ACEA
RT-CES system at different time periods along the growth and
differentiative curve of ES-derived cardiomyocytes seeded in ACEA
E-PLATES. Each well of the E-PLATE comprises an impedance
measurement electrode array, comprising two circle-on-line
electrode structures. FIGS. 5A, 5B and 5C show the cell index
curves at time resolution of 40 milli-seconds for time points at
24, 48 and 72 hrs after cell seeding, respectively. FIG. 5D shows
the cell index curves monitored at 1 hr time resolution.
FIGS. 6A and 6B show the cell index curve for ES-derived
cardiomyocytes and corresponding beat rate as analyzed using
Fourier transform.
FIG. 7 shows the detection of cardiomyocyte beating using the ACEA
RT-CES system for the mouse ES-derived cardiomyocytes treated with
4.4 uM sotalol. FIGS. 7A and 7B show the beating rate and
amplitude, respectively, for the cells before and after the
treatment. FIG. 7C shows overall cell index curves whilst FIG. 7D
shows the cell-index based beating curve for a short time period.
Note that one second displayed in FIGS. 7C and 7D is equivalent to
40 milli-seconds for real time measurement. The time resolution of
the RT-CES measurement for FIGS. 7C and 7D is 40 milli-seconds.
FIGS. 8A and 8B show the detection of cardiomyocyte beating using
the ACEA RT-CES system to assess the effect of chemical compound
carbachol (333 nM, 8A) and isoproternaol (4.4 uM, 8B) on the
beating of cardiomyocyte. Note that one second displayed in FIGS.
8A and 8B is equivalent to 40 milli-seconds for real time
measurement. The time resolution of the RT-CES measurement for
FIGS. 8A and 8B is 40 milli-seconds.
FIGS. 9A-S show the detection of cardiomypcyte beating using the
ACEA RT-CES system to assess the effect of various chemical
compounds on the beating of mouse ES-derived cardiomyocytes: (9A)
33.3 uM sotalol with 0.033% DMSO; (9B) 400 nM Astemizole with
0.004% DMSO; (9C) 200 nM terfenadine with 0.065% DMSO; (9D) 13.3 uM
Erythromycin with 0.13% DMSO; (9E) 20 uM moxifloxacin with 0.3%
DMSO; (9F) 20 uM pentamidine with 0.2% DMSO; (9G) 4.4 uM
amitripyline with 0.04% DMSO; (9H) 130 nM verapamil with 0.0013%
DMSO; (9I) 13.3 uM rosiglitazone with 0.13% DMSO; (9J) 13.3 uM
rofecoxib; (9K) 4.4 uM celecoxib; (9L) 40 uM doxorubicin with 0.4%
DMSO; (9M) 13.3 uM cyclosporine with 0.13% DMSO; (9N) 4.4 uM
propranolol with 0.04% DMSO; (9O) 9.1 nM E4031; (9P) 8 uM DDT with
0.08% DMSO; (9Q) 8 uM PCB with 0.08% DMSO; (9R) 8 uM endosulfan
with 0.08% DMSO; (9S) 0.13% DMSO. FIG. 9S shows the 0.13% DMSO
control, indicating that DMSO at such a concentration does not
produce a significant change on the beating of the cardiomyocytes.
Note that one second displayed in FIGS. 9A-9S is equivalent to 40
milli-seconds for real time measurement. The time resolution of the
measurement for FIGS. 9A-9S is 40 milli-seconds.
FIG. 10 shows the change of the beating pattern of mouse ES-derived
cardiomyocytes as a result of treatment with different compounds as
monitored by ACEA RT-CES system. The compounds being tested here
include 130 nM verapamil, 40 nM astemizole, 20 uM sotalol; 200 nM
trefenadine; 4 uM propranolol; 4 uM celexocib; 150 nM carbachol and
82 nM E4031.
FIG. 11 shows the change of the beating pattern of mouse ES-derived
cardiomyocytes as a result of treatment with compound Aztemizole as
monitored by ACEA RT-CES system. The compounds concentrations being
tested here include 400 nM, 40 nM, 13.3 nM, 1.33 nM, 0.133 nM and
40 .mu.M.
FIG. 12 shows a pair of extracellular recording electrodes,
comprising a recording electrode structure on the substrate and a
reference electrode external to the substrate.
FIG. 13 shows examples of electrode structures having multiple
electrode elements that can be used for extracellular recording
and/or impedance measurement where 13A, 13B, 13C, 13D, 13E and 13F
are the rectangular shape, circle-on-line shape, a complete
interdigitated electrode array, sinuosoidal shape, castellated
shapes and a complete circle-on-line electrode array,
respectively.
FIGS. 14A and 14B show two examples of extracellular recording
devices where each well comprises one recording electrode structure
comprising multiple electrode elements and a reference electrode
having large surface area.
FIG. 15 shows an example of an extacellular recording device where
each well of the device comprises a simple, circle-shaped recording
electrode and a reference electrode (1501) having large surface
area (1502).
FIG. 16 shows an example of a device of the present invention where
in one well, a pair of impedance measurement electrodes is located
on the substrate and one of the impedance measurement electrode
structures is used as an extracellular recording electrode,
together with an externally applied reference electrode.
FIG. 17 shows a multi-electrode chip comprising an array of 30
electrodes where the electrode diameter is 50 .mu.m and the
interval between each two neighbor electrode tips is 50 .mu.m.
FIG. 18 shows the extracellular field potential (FP) recorded for
mouse ES-derived cardiomyocytes on day 2 and day 3 after cell
plating using electrode array shown in FIG. 17.
FIG. 19 shows the extracellular field potential (FP) recorded for
mouse ES-derived cardiomyocytes on day 3 after cell plating using
electrode array shown in FIG. 17. The cells are treated with
compound E4031 at different concentrations ranging form 0 nM
(control), 75 nM, 150 nM, 250 nM and 750 nM respectively.
FIG. 20 shows the dependency of field potential duration in
milli-seconds on the concentration of E4031.
FIG. 21 shows the "anti-arrhythmic" effect of compound E4031 at 75
nM and 150 nM on field potential frequency for the mouse ES-cell
derived cardiomyocytes.
FIG. 22 shows extra-cellular field potentials recorded for mouse
stem-cell derived cardiomyocytes obtained using a device of the
present invention, where the recording electrode is a
circle-on-line electrode structure and the reference electrode is a
gold wire electrode that is introduced into the well after cell
seeding. FIGS. 22A, 22B, 22C and 22D shows the recorded waveforms
before treatment, .about.18 seconds, .about.3 minutes and 5.5
minutes after treatment with E4031, respectively.
FIG. 23 shows extra-cellular field potentials recorded for mouse
stem-cell derived cardiomyocytes obtained using a device of the
present invention, where the recording electrode is a
circle-on-line electrode structure and the reference electrode is a
gold wire electrode that is introduced into the well after cell
seeding. FIGS. 23A, 23B, 23C, 23 D and 23E show the recorded
waveforms before treatment, 10 seconds, 50 seconds, 3 minutes and 9
minutes after treatment with 3 uM Quinidine, respectively.
FIG. 24 shows parallel impedance monitoring of beating of mouse
stem-cell derived cardiomyocytes obtained using a device of the
present invention, where the impedance is monitored using an
electrode array comprising two electrode structures each of which
comprises multiple circle-on-line electrode elements. FIGS. 24A,
24B, 24C, and 24 D show the recorded impedance beating signals
before treatment, 1.5 minute, .about.3 minute and .about.11 minute
after treatment with 3 uM Quinidine, respectively.
FIG. 25 shows extra-cellular field potentials recorded for primary
cardiomyocytes obtained using a device of the present invention,
where the recording electrode is a circle-on-line electrode
structure and the reference electrode is a gold wire electrode that
is introduced into the well after cell seeding.
FIG. 26 shows a device of the present invention wherein the well
(2605) of the device comprises a recording electrode (2601) for
extra-cellular recording, an interdigitated electrode array (2602)
comprising two electrode structures (2603 and 2606) each of which
comprises multiple electrode elements. The electrodes are
positioned on non-conductive substrate 2604. Extracellular
recording of field potentials can be achieved by amplifying and
recording voltage signals between the recording electrode 2601 and
one electrode structure 2603 (or 2606). The impedance measurement
can be conducted by monitoring impedance between electrode
structures 2603 and 2606.
FIG. 27 shows the design of an electrode array used for combined
cell-impedance measurement and extra-cellular recording.
FIG. 28 shows the impedance beating result obtained for the Cor. At
cells (mouse ES derived cardiomyocytes) for .about.12 seconds with
time-solution about 28.8 ms per data point. The impedance data is
represented with cell index, a dimension less parameter.
FIG. 29 shows the extracellular field potential signals obtained
for the Cor. At cells (mouse ES derived cardiomyocytes) using the
electrode array in FIG. 27 for three different recording modes,
including: (1) ECR electrode versus impedance electrode-structure,
(2) ECR electrode versus external, gold, reference electrode and
(3) impedance electrode-structure versus external, gold, reference
electrode.
FIG. 30 shows the design of another electrode array used for
combined cell-impedance measurement and extra-cellular
recording.
FIG. 31 shows the impedance beating result obtained for the Cor. At
cells (mouse ES derived cardiomyocytes) for .about.12 seconds with
time-solution about 28.8 ms per data point. The impedance data is
represented with cell index, a dimension less parameter.
FIG. 32 shows the extracellular field potential signals obtained
for the Cor. At cells (mouse ES derived cardiomyocytes) using the
electrode array in FIG. 20 for three different recording modes,
including: (1) ECR electrode versus impedance electrode-structure,
(2) ECR electrode versus external, gold, reference electrode and
(3) impedance electrode-structure versus external, gold, reference
electrode.
FIG. 33 shows top view and cross-sectional view of a device of the
present invention.
FIG. 34 shows a device of the present invention where the well of
the device 3405 comprises a recording electrode 3401 and an
interdigitated electrode array 3406 on the substrate 3404, and an
external, reference electrode 3402.
DETAILED DESCRIPTION OF THE INVENTION
A. Definitions
For clarity of disclosure, and not by way of limitation, the
detailed description of the invention is divided into the
subsections that follow. Further, unless defined otherwise, all
technical and scientific terms used herein have the same meaning as
is commonly understood by one of ordinary skill in the art to which
this invention belongs. All patents, applications, published
applications and other publications referred to herein are
incorporated by reference in their entirety. If a definition set
forth in this section is contrary to or otherwise inconsistent with
a definition set forth in the patents, applications, published
applications and other publications that are herein incorporated by
reference, the definition set forth in this section prevails over
the definition that is incorporated herein by reference.
As used herein, "biocompatible membrane" means a membrane that does
not have deleterious effects on cells, including the viability,
attachment, spreading, motility, growth, cell division or cell
beating.
A "biomolecular coating" or a "biological molecule coating" is a
coating on a surface that comprises a molecule that is a naturally
occurring biological molecule or biochemical, or a biochemical
derived from or based on one or more naturally occurring
biomolecules or biochemicals. For example, a biological molecule
coating can include an extracellular matrix component (e.g.,
fibronectin, collagens), or a derivative thereof, or can comprise a
biochemical such as polylysine or polyornithine, which are
polymeric molecules based on the naturally occurring biochemicals
lysine and ornithine. Polymeric molecules based on naturally
occurring biochemicals such as amino acids can use isomers or
enantiomers of the naturally-occurring biochemicals.
A "cell-free zone" is a region of the device where cells, when
added to the device, are not physically present. The "cell-free
zone" may be a region on the substrate, which is blocked from cell
access. The "cell-free zone" may be a spatial position free from
contact with the substrate such as a spatial position above a
substrate base and within the volume of the well. The "cell-free
zone" should permit the passage of electrical current such as
through a conductive medium. For example, a reference electrode or
external electrode may be suspended in a cell-free zone, free from
contact with the substrate, yet remain electrically coupled to an
extracellular recording electrode disposed on the substrate.
A "cell-free electrode" is an electrode that is free from contact
with all cells present in the device.
The term "free from contact" or "free from direct contact" refers
to two individuals, whether cells or apparatus components that lack
contacting surfaces. A cell suspended in medium, in which only the
medium contacts an electrode is "free from contact" or "free from
direct contact" with the electrode.
An "organic compound coating" is a coating on a surface that
includes an organic compound. For example an organic compound may
include a natural ligand or an agonist or an antagonist for a cell
surface receptor.
An "extracellular matrix component" is a molecule that occurs in
the extracellular matrix of an animal. It can be a component of an
extracellular matrix from any species and from any tissue type.
Nonlimiting examples of extracellular matrix components include
laminins, collagens fibronectins, other glycoproteins, peptides,
glycosaminoglycans, proteoglycans, etc. Extracellular matrix
components can also include growth factors.
An "electrode" is a structure having a high electrical
conductivity, that is, an electrical conductivity much higher than
the electrical conductivity of the surrounding materials, which in
the present invention are typically nonconductive. An
"extracellular recording electrode" or "recording electrode" or
"ECR electrode" is such a structure used to detect electrical
signal corresponding to extracellular field potential of the cell
or cell population. For instance, a "recording electrode" may be
used to monitor the extracellular field potential of a
cardiomyocyte during the generation of membrane action potentials.
A "reference electrode" is the complementary structure used to
complete the electrical circuit during extracellular recording. An
"impedance electrode" or an "impedance measurement electrode" or
"impedance measurement electrode structure" is a structure, such as
an electrode, used for impedance monitoring. An "impedance
electrode" may also operate as an extracellular recording electrode
and thus may provide both impedance monitoring and extracellular
recording measurements, albeit at different time points.
As used herein, an "electrode structure" refers to a single
electrode, particularly one with a complex structure (as, for
example, a spiral electrode structure), or a collection of at least
two electrode elements that are electrically connected together.
All the electrode elements within an "electrode structure" are
electrically connected.
As used herein, "electrode element" refers to a single structural
feature of an electrode structure, such as, for example, a
fingerlike or branched projection of an interdigitated electrode
structure. An electrode structure may have a plurality of electrode
elements.
As used herein, a "unitary electrode structure" refers to a single
electrode that is unbranched. That is, a "unitary electrode
structure" does not include a plurality of electrode elements. For
example, an unitary electrode structure may be of a circle, a
square or other geometry.
As used herein, an "electrode array" or "electrode structure unit"
is two or more electrode structures that are constructed to have
dimensions and spacing such that they can, when connected to a
signal source, operate as a unit to generate an electrical field in
the region of spaces around the electrode structures. Preferred
electrode structure units of the present invention can measure
impedance changes due to cell attachment to an electrode surface.
Non-limiting examples of electrode structure units are
interdigitated electrode structure units and concentric electrode
structure units.
An "electrode bus" is a portion of an electrode that connects
individual electrode elements or substructures. An electrode bus
provides a common conduction path from individual electrode
elements or individual electrode substructures to another
electrical connection. In the devices of the present invention, an
electrode bus can contact each electrode element of an electrode
structure and provide an electrical connection path to electrical
traces that lead to a connection pad.
"Electrode traces" or "electrically conductive traces" or
"electrical traces", are electrically conductive paths that extend
from electrodes or electrode elements or electrode structures
toward one end or boundary of a device or apparatus for connecting
the electrodes or electrode elements or electrode structures to an
analyzer or amplifier, such as an impedance amplifier, a voltage
amplifier and the like. Both impedance electrodes and extracellular
recording electrodes may connect to an "electrode trace." The end
or boundary of a device may correspond to the connection pads on
the device or apparatus.
A "connection pad" is an area on an apparatus or a device of the
present invention which is electrically connected to at least one
electrode or all electrode elements within at least one electrode
structure on an apparatus or a device and which can be operatively
connected to external electrical circuits (e.g., an impedance
measurement circuit or a signal source or an extracellular voltage
signal amplifier). The electrical connection between a connection
pad and an impedance measurement circuit, an extracellular
recording circuit or a signal source can be direct or indirect,
through any appropriate electrical conduction means such as leads
or wires. Such electrical conduction means may also go through
electrode or electrical conduction paths located on other regions
of the apparatus or device.
"Interdigitated" means having projections coming one direction that
interlace with projections coming from a different direction in the
manner of the fingers of folded hands (with the caveat that
interdigitated electrode elements preferably do not contact one
another).
As used herein, a "high probability of contacting an electrode
element" means that, if a cell is randomly positioned within the
sensor area of a device or apparatus of the present invention, the
probability of a cell (or particle) contacting an electrode
element, calculated from the average diameter of a cell used on or
in a device or apparatus of the present invention, the sizes of the
electrode elements, and the size of the gaps between electrode
elements, is greater than about 50%, more preferably greater than
about 60%, yet more preferably greater than about 70%, and even
more preferably greater than about 80%, greater than about 90%, or
greater than about 95%.
As used herein, "at least two electrodes fabricated on the
substrate" means that the at least two electrodes are fabricated or
made or produced on the substrate. The at least two electrodes can
be on the same side of the substrate or on the different side of
the substrate. The substrate may have multiple layers, the at least
two electrodes can be either on the same or on the different layers
of the substrate.
As used herein, "at least two electrodes fabricated to a same plane
of the substrate" means that, if the nonconducting substrate has
multiple layers, the at least two electrodes are fabricated to the
same layer of the substrate.
As used herein, "an electrode positioned on a different plane"
refers to the positioning of an electrode, typically an external
electrode or reference electrode, above, below or along a different
surface angle than that which it is compared. An "electrode
positioned on a different plane" may be parallel to that of the
first.
As used herein, "the . . . electrodes (or electrode structures)
have substantially the same surface area" means that the surface
areas of the electrodes referred to are not substantially different
from each other, so that the impedance change due to cell
attachment or growth on any one of the electrodes (or electrode
structures) referred to will contribute to the overall detectable
change in impedance to a same or similar degree as the impedance
change due to cell attachment or growth on any other of the
electrodes (or electrode structures) referred to. In other words,
where electrodes (or electrode structures) have substantially the
same surface area, any one of the electrodes can contribute to
overall change in impedance upon cell attachment or growth on the
electrode. In most cases, the ratio of surface area between the
largest electrode and the smallest electrode that have
"substantially the same surface area" is less than 10. Preferably,
the ratio of surface area between the largest electrode and the
smallest electrode of an electrode array is less than 5, 4, 3, 2,
1.5, 1.2 or 1.1. More preferably, the at least two electrodes of an
electrode structure have nearly identical or identical surface
area.
As used herein, "the device has a surface suitable for cell
attachment or growth" means that the electrode and/or non-electrode
area of the apparatus has appropriate physical, chemical or
biological properties such that cells of interest can viably attach
on the surface and new cells can continue to attach, while the cell
culture grows, on the surface of the apparatus. However, it is not
necessary that the device, or the surface thereof, contain
substances necessary for cell viability or growth. These necessary
substances, e.g., nutrients or growth factors, can be supplied in a
medium. Preferably, when a suspension of viable cardiomyocytes,
neuron cells, muscle cells or other excitable cells or other
adherent cells such as epithelial cells or endothelial cells is
added to the "surface suitable for cell attachment" when at least
50% of the cells are adhering to the surface within twelve hours.
More preferably, a surface that is suitable for cell attachment has
surface properties so that at least 70% of the cells are adhering
to the surface within twelve hours of plating (i.e., adding cells
to the chamber or well that comprises the said device). Even more
preferably, the surface properties of a surface that is suitable
for cell attachment results in at least 90% of the cells adhering
to the surface within twelve hours of plating. Most preferably, the
surface properties of a surface that is suitable for cell
attachment results in at least 90% of the cells adhering to the
surface within eight, six, four, two hours of plating.
As used herein, "detectable change in impedance between or among
said electrodes" (or "detectable change in impedance between or
among the electrode structures") means that the impedance between
or among the electrodes (or electrode structures) would have a
significant change that can be detected by an impedance analyzer or
impedance measurement circuit when cells attach on the electrode
surfaces. The impedance change refers to the difference in
impedance values when cells are attached to the electrode surface
and when cells are not attached to the electrode surface, or when
the number, type, activity, adhesiveness, or morphology of cells
attached to the electrode-comprising surface of the apparatus
changes. In most cases, the change in impedance is larger than 0.1%
to be detectable. Preferably, the detectable change in impedance is
larger than 1%, 2%, 5%, or 8%. More preferably, the detectable
change in impedance is larger than 10%. Impedance between or among
electrodes is typically a function of the frequency of the applied
electric field for measurement. "Detectable change in impedance
between or among the electrodes" does not require the impedance
change at all frequencies being detectable. "Detectable change in
impedance between or among said electrodes" only requires a
detectable change in impedance at any single frequency (or multiple
frequencies). In addition, impedance has two components, resistance
and reactance (reactance can be divided into two categories,
capacitive reactance and inductive reactance). "Detectable change
in impedance between or among said electrodes" requires only that
either one of resistance and reactance has a detectable change at
any single frequency or multiple frequencies. In the present
application, impedance is the electrical or electronic impedance.
The method for the measurement of such impedance is achieved by,
(1) applying a voltage between or among the electrodes at a given
frequency (or multiple frequencies, or having specific voltage
waveform) and monitoring the electrical current through said
electrodes at the frequency (or multiple frequencies, or having
specific waveform), dividing the voltage amplitude value by the
current amplitude value to derive the impedance value; (2) applying
an electric current of a single frequency component (or multiple
frequencies or having specific current wave form) through said
electrodes and monitoring the voltage resulted between or among
said electrodes at the frequency (or multiple frequencies, or
having specific waveform), dividing the voltage amplitude value by
the current amplitude value to derive the impedance value; (3)
other methods that can measure or determine electric impedance.
Note that in the description above of "dividing the voltage
amplitude value by the current amplitude value to derive the
impedance value", the "division" is done for the values of current
amplitude and voltage amplitude at same frequencies. Measurement of
such electric impedance is an electronic or electrical process that
does not involve the use of any reagents.
As used herein, "multiple pairs of electrodes or electrode
structures spatially arranged according to wells of a multi-well
microplate" means that the multiple pairs of electrodes or
electrode structures of a device or apparatus are spatially
arranged to match the spatial configuration of wells of a
multi-well microplate so that, when desirable, the device can be
inserted into, joined with, or attached to a multiwell plate (for
example, a bottomless multiwell plate) such that multiple wells of
the multi-well microplate will comprise electrodes or electrode
structures.
As used herein, "arranged in a row-column configuration" means
that, in terms of electric connection, the position of an
electrode, an electrode array or a switching circuit is identified
by both a row position number and a column position number.
As used herein, "each well contains substantially same number . . .
of cells" means that the lowest number of cells in a well is at
least 50% of the highest number of cells in a well. Preferably, the
lowest number of cells in a well is at least 60%, 70%, 80%, 90%,
95% or 99% of the highest number of cells in a well. More
preferably, each well contains an identical number of cells.
As used herein, "each well contains . . . same type of cells" means
that, for the intended purpose, each well contains same type of
cells; it is not necessary that each well contains exactly
identical type of cells. For example, if the intended purpose is
that each well contains mammalian cells, it is permissible if each
well contains same type of mammalian cells, e.g., human cells, or
different mammalian cells, e.g., human cells as well as other
non-human mammalian cells such as mice, goat or monkey cells,
etc.
As used herein, "each well contains . . . serially different
concentration of a test compound" means that each well contains a
test compound with a serially diluted concentrations, e.g., an
one-tenth serially diluted concentrations of 1 M, 0.1 M, 0.01 M,
etc.
As used herein, "dose-response curve" means the dependent
relationship of response of cells on the dose concentration of a
test compound. The response of cells can be measured by many
different parameters. For example, a test compound is suspected to
have cytotoxicity and cause cell death. Then the response of cells
can be measured by percentage of non-viable (or viable) cells after
the cells are treated by the test compound. Plotting this
percentage of non-viable (or viable) cells as a function of the
dose concentration of the test compound constructs a dose response
curve. In the present application, the percentage of non-viable (or
viable) cells can be expressed in terms of measured impedance
values, or in terms of cell index derived from impedance
measurement, or in terms of cell change indexes. For example, for a
give cell type and under specific cellular physiological condition
(e.g., a particular cell culture medium), cell index can be shown
to have a linear correlation or positive correlation with the
number of viable cells in a well from which cell index was derived
from the impedance measurement. Thus, in the present application,
one can plot cell index as a function of the dose concentration of
the test compound to construct a "dose-response curve". Note that,
generally, cell index not only correlate with the number of viable
cells in the wells but also relate to the cell morphology and cell
attachment. Thus plotting cell index versus dose concentration
provides information not only about number of cells but also about
their physiological status (e.g. cell morphology and cell
adhesion). Furthermore, an important advantage offered by the
system and devices of the present invention is that in a single
experiment, one can obtain "dose-response curves" at multiple time
points since the system allows for the continuous monitoring of
cells and provides impedance measurement at many time points over a
time range as short as a few minutes to as long as days or
weeks.
As used herein, "microelectrode strip or electrode strip" means
that a non-conducting substrate strip on which electrodes or
electrode structure units are fabricated or incorporated. The
non-limiting examples of the non-conducting substrate strips
include polymer membrane, glass, plastic sheets, ceramics,
insulator-on-semiconductor, fiber glass (like those for
manufacturing printed-circuits-board). Electrode structure units
having different geometries can be fabricated or made on the
substrate strip by any suitable microfabrication, micromachining,
or other methods. Non-limiting examples of electrode geometries
include interdigitated electrodes, circle-on-line electrodes,
diamond-on-line electrodes, castellated electrodes, spiral
electrodes or sinusoidal electrodes. Characteristic dimensions of
these electrode geometries may vary from as small as less than 5
micron, or less than 10 micron, to as large as over 200 micron,
over 500 micron, over 1 mm. The characteristic dimensions of the
electrode geometries refer to the smallest width of the electrode
elements, or smallest gaps between the adjacent electrode elements,
or size of a repeating feature on the electrode geometries. The
microelectrode strip can be of any geometry for the present
invention. One exemplary geometry for the microelectrode strips is
rectangular shape--having the width of the strip between less than
50 micron to over 10 mm, and having the length of the strip between
less than 60 micron to over 15 mm. An exemplary geometry of the
microelectrode strips may have a geometry having a width of 200
micron and a length of 20 mm. A single microelectrode strip may
have two electrodes serving as a measurement unit, or multiple such
two-electrodes serving as multiple measurement units, or a single
electrode structure unit as a measurement unit, or multiple
electrode structure units serving as multiple electrode structure
units. In one exemplary embodiment, when multiple electrode
structure units are fabricated on a single microelectrode strip,
these electrode structure units are positioned along the length
direction of the strip. The electrode structure units may be of
squared-shape, or rectangular-shape, or circle shapes. Each of
electrode structure units may occupy size from less than 50 micron
by 50 micron, to larger than 2 mm.times.2 mm.
As used herein, "sample" refers to anything which may contain a
moiety to be isolated, manipulated, measured, quantified, detected
or analyzed using apparatuses, microplates or methods in the
present application. The sample may be a biological sample, such as
a biological fluid or a biological tissue. Examples of biological
fluids include suspension of cells in a medium such as cell culture
medium, urine, blood, plasma, serum, saliva, semen, stool, sputum,
cerebral spinal fluid, tears, mucus, amniotic fluid or the like.
Biological tissues are aggregates of cells, usually of a particular
kind together with their intercellular substance that form one of
the structural materials of a human, animal, plant, bacterial,
fungal or viral structure, including connective, epithelium, muscle
and nerve tissues. Examples of biological tissues also include
organs, tumors, lymph nodes, arteries and individual cell(s). The
biological samples may further include cell suspensions, solutions
containing biological molecules (e.g. proteins, enzymes, nucleic
acids, carbohydrates, chemical molecules binding to biological
molecules).
As used herein, a "liquid (fluid) sample" refers to a sample that
naturally exists as a liquid or fluid, e.g., a biological fluid. A
"liquid sample" also refers to a sample that naturally exists in a
non-liquid status, e.g., solid or gas, but is prepared as a liquid,
fluid, solution or suspension containing the solid or gas sample
material. For example, a liquid sample can encompass a liquid,
fluid, solution or suspension containing a biological tissue.
A "compound" or "test compound" is any compound whose activity or
direct or indirect effect or effects on cells is investigated in
any assay. A test compound can be any compound, including, but not
limited to, a small molecule, a large molecule, a molecular
complex, an organic molecule, an inorganic molecule, a biomolecule
or biological molecule such as but not limited to a lipid, a
steroid, a carbohydrate, a fatty acid, an amino acid, a peptide, a
protein, a nucleic acid, or any combination thereof. A test
compound can be a synthetic compound, a naturally occurring
compound, a derivative of a naturally-occurring compound, etc. The
structure of a test compound can be known or unknown. In one
application of the present invention, a compound is capable of, or
is suspected of, effecting cell adhesion or cell spreading. In
another application of present invention, a compound is capable of,
or is suspected of, stimulating or inhibiting cell adhesion or cell
spreading. In still another application, a compound is capable of,
or is suspected of, interacting with cells (for example, binding to
cell surface receptor, or inhibiting certain intracellular signal
transduction pathway, or activating cells).
A "known compound" is a compound for which at least one activity is
known. In the present invention, a known compound preferably is a
compound for which one or more direct or indirect effects on cells
is known. Preferably, the structure of a known compound is known,
but this need not be the case. Preferably, the mechanism of action
of a known compound on cells is known, for example, the effect or
effects of a known compound on cells can be, as nonlimiting
examples, effects on cell beating, cell viability, cell adhesion,
apoptosis, cell differentiation, cell proliferation, cell
morphology, cell cycle, IgE-mediated cell activation or
stimulation, receptor-ligand binding, cell number, cell quality,
cell cycling, cell adhesion, cell spreading, etc.
An "impedance value" is the impedance measured for electrodes in a
well with or without cells present. Impedance is generally a
function of the frequency, i.e., impedance values depend on
frequencies at which the measurement was conducted. For the present
application, impedance value refers to impedance measured at either
single frequency or multiple frequencies. Furthermore, impedance
has two components, one resistance component and one reactance
component. Impedance value in the present application refers to
resistance component, or reactance component, or both resistance
and reactance component. Thus, when "impedance value" was measured
or monitored, we are referring to that, resistance, or reactance,
or both resistance and reactance were measured or monitored. In
many embodiments of the methods of the present application,
impedance values also refer to parameter values that are derived
from raw, measured impedance data. For example, cell index, or
normalized cell index, or delta cell index could be used to
represent impedance values.
A "Cell Index" or "CI" is a parameter that can derived from
measured impedance values and that can be used to reflect the
change in impedance values. There are a number of methods to derive
or calculate Cell Index. The details of the method for calculating
Cell Index, Normalized Cell Index, Delta Cell Index and cell change
index can be found in U.S. patent application Ser. No. 10/705,447,
filed on Nov. 10, 2003; U.S. patent application Ser. No.
10/705,615, filed on Nov. 10, 2003; U.S. patent application Ser.
No. 10/987,73, filed on Nov. 12, 2004; U.S. patent application Ser.
No. 11/055,639, filed on Feb. 9, 2005; U.S. patent application Ser.
No. 11/198,831, filed on Aug. 4, 2005; U.S. patent application Ser.
No. 11/197,994, filed on Aug. 4, 2005; U.S. patent application Ser.
No. 11/235,938, filed on Sep. 27, 2005, all of them are
incorporated here by reference.
A "Normalized Cell Index" at a given time point is calculated by
dividing the Cell Index at the time point by the Cell Index at a
reference time point. Thus, the Normalized Cell Index is 1 at the
reference time point. Generally, for an assay involving treatment
of the cells with compounds or with other bio-manipulation of the
cells, the reference time point is the last time point for
impedance measurement before the treatment of the cells.
A "delta cell index" at a given time point is calculated by
subtracting the cell index at a standard time point from the cell
index at the given time point. Thus, the delta cell index is the
absolute change in the cell index from an initial time (the
standard time point) to the measurement time.
A "Cell Change Index" or "CCI" is a parameter derived from Cell
Index and "CCI" at a time point is equal to the 1.sup.St order
derive of the Cell Index with respect to time, divided by the Cell
Index at the time point. In other words, CCI is calculated as
.function..function..function. ##EQU00001##
The term "extracellular recording" refers to measuring, monitoring
and/or recording of electric potential difference between two
electrodes typically caused by ionic movement or ionic current
through the media or solution due to charge fluctuations across ion
channels in a cell or in a group of cells. The cells are in the
media or the solution. In contrast to intracellular recording where
the recording electrodes are placed inside a cell through the cell
membrane, the extracellular recording electrodes are located
outside of the cells.
B. Method to Assess and Quantify Cardiomyocyte Viability In Vitro
Using Cell Sensor Impedance Technology
The methods and devices of the present invention may assess or
quantify the viability of cardiomyocyte or cardiomyocyte precursor
cells in addition to performing extracellular recording assays.
Such assessment may be performed prior to performing an
extracellular recording assay to assess the initial viability or
characteristics of cells or may be performed periodically
throughout the extracellular recording process to continually
monitor the viability or characteristics of cells, or may be
performed in parallel with extracellular recording.
Isolated primary cardiomyocytes, ES-derived cardiomyocytes and
adult stem cell-derived cardiomyocytes can be maintained in
culture. These cells provide an excellent model system to study the
effect of drugs and other factors on cardiomyocyte viability.
Exemplary steps involved in using an impedance-monitoring system
for measurement of cardiomyocyte viability include: (1) Provide a
single-well or multi-well device that comprise microelectrode
arrays in well(s) of the device, which can be used for monitoring
cell-substrate impedance. (2) Optionally coat wells of the device
with either fibronectin or other matrix proteins. (3) Seed either
embryonic stem cells (ES cells) of mammalian origin, mammalian
adult stem cell-derived cardiomyocytes or primary cardiomyocytes
isolated directly from mammalian heart tissue at specific seeding
densities to the wells of the device. (4) Allow the cells to attach
and spread. (5) Monitor cardiomyocyte viability over time using the
impedance-monitoring system to monitor electrode impedance at
pre-specified intervals of time (non-limiting examples of the time
include 5, 15, 30 minutes, 1 hr and 2 hrs) for specified length of
time such as 12, 24, 48, 72 hours or longer.
One example of the impedance measurement system is ACEA
Biosciences' RT-CES system, where the device is ACEA E-PLATE in the
form of microtiter plates whose wells comprise microelectrode
structures. Thus in a related embodiment, exemplary steps involved
in using the RT-CES system for measurement of cardiomyocyte
viability include: (1) Optionally coat E-PLATES with either
fibronectin or other matrix proteins. (2) Seed either embryonic
stem cells (ES cells) of mammalian origin, mammalian adult stem
cell-derived cardiomyocytes or primary cardiomyocytes isolated
directly from mammalian heart tissue at specific seeding densities
to the wells of the device. (3) Allow the cells to attach and
spread. (4) Monitor cardiomyocyte viability over time using the
impedance-monitoring system to monitor electrode impedance at
pre-specified intervals of time (non-limiting examples of the time
include 5, 15, 30 minutes, 1 hr and 2 hrs) for specified length of
time such as 12, 24, 35, 48, 72 hours or longer.
As an example, we describe here the use of the ACEA RT-CES system
to measure and monitor the attachment, growth and viability of
mouse ES-derived cardiomyocytes which were seeded at different
seeding densities. Mouse ES cells were seeded at a density ranging
from 3000 cells to 50,000 cells per well in ACEA E-PLATES that had
been precoated with fibronectin. The attachment, growth and
viability of the cells were monitored on the RT-CES system
measuring impedance signal in the form of cell index every 30
minutes for 48 hours. At about 48 hrs after cell seeding, the
growth of the cells had ceased and the appearance of beating
cardiomyocytes were evident as judged by looking at the cells
inside the E-PLATE under the microscope. As such, these cells would
be suitable for use, for example, in extracellular recording
experiments to determine the effect of external stimuli, such as
changes in cardiomyocyte beating after administration of a test
compound, or in the experiments to monitor cardiomyocyte beating
through impedance measurement at milli-second time resolution. FIG.
1 shows the cell index curves measured on RT-CES system for 4
different seeding densities (3750, 7500, 15,000 and 30,000 cells
per well) of mouse ES-derived cardiomyocytes. For such long term
measurement, cell electrode impedance and corresponding cell
indices were measured at about 15 minute intervals. Based on the
cell index growth and viability curves, it is evident that the
extent of the impedance signal correlates well with the seeding
density of viable ES-derived cardiomyocytes.
C. Method to Assess and Quantify Modulation of Cardiomyocyte
Viability In Vitro Using Cell Sensor Impedance Technology
Certain cardiotoxic drugs can directly affect the viability of
cardiomyocytes. Accordingly, continuing to monitor the viability of
cardiomyocytes over time may be used to supplement data obtained
from extracellular recording of cells. Exemplary steps involved in
using an impedance-measurement system for measurement of loss of
viability of cardiomyocyte include: (1) Optionally coat E-PLATES
with either fibronectin or other matrix proteins. (2) Seed either
embryonic stem cells (ES cells) of mammalian origin, mammalian
adult stem cell-derived cardiomyocytes or primary cardiomyocytes
isolated directly from mammalian heart tissue at specific seeding
densities to the wells of the device. (3) Allow the cells to attach
and spread. (4) Monitor cardiomyocyte viability over time using the
impedance-monitoring system to monitor electrode impedance at
pre-specified intervals of time for specified length of time such
as 12, 24, 37, 48, 72 hours or longer (5) At certain time after
cell seeding, treat the cell with cytotoxic agent at one or more
concentration; using the vehicle that the agent is dissolved in as
a control. (6) Continue monitoring the cardiomyocytes at
pre-specified intervals of time for specified length of time such
as additional 12, 24, 48, 72 hours or longer (7) Quantify the
extent of cardiotoxicity by normalizing the cell index values
immediately prior to agent addition and determine the normalized
cell index at a given time point after agent addition;
alternatively the rate of cytotoxicity can also be quantified for a
given time period after compound addition for a given agent
concentration or a group of concentrations. The extent of
cytotoxicity can be expressed as IC-50 value which quantifies the
activity of the agent with respect to the cardiomyocytes.
As an example, we describe here the use of the ACEA RT-CES system
to measure and monitor the attachment and growth of mouse ES cells
derived cardiomyocytes and subsequently treated with a cytotoxic
agent (FIG. 2). Mouse ES-derived cardiomyocytes were seeded at a
density of 25,000 cells per well in ACEA E-Plates that had been
precoated with fibronectin. The attachment and growth of the cells
were monitored on RT-CES system for 72 hours and then treated with
increasing doses of the compound of sodium dichromate dehydrate
which is known to induce cytotoxicity.
According to FIG. 2A of the plot of normalized cell index for cells
in different wells treated with different concentrations of sodium
dichromate dehydrate (SDD), SDD causes a concentration dependent
decrease in viability of ES-derived cardiomyocytes. To quantify the
extent of sodium dichromate dehydrate activity against the
cardiomyocytes, the normalized cell indices at 24 hrs after
compound treatment were plotted against the log of the
corresponding sodium dichromate dehydrate concentrations. From the
sigmoidal curve, shown in FIG. 2B, half maximal activity or IC-50
value of 1.48 uM was derived for the compound.
D. Method to Assess and Quantify Modulation of Cardiomyocyte
Morphology In Vitro Using Cell Sensor Impedance Technology
Certain cardiotoxic drugs can illicit their effect by affecting the
morphological aspects of cardiomyocyte morphology. For example, it
is well known that compounds such as b-2 adrenergic receptor
agonists can induce morphological changes resulting in an elongated
cardiomyocyte morphology, otherwise known as hypertrophy.
Morphological changes can occur immediately in the order of minutes
as with certain GPCR agonists or can be of longer duration
detectable over several days. The time resolution of the RT-CES
system can be used to distinguish between different kinds of
morphological effects. The steps involved in using an
impedance-monitoring system on the RT-CES system for measurement of
morphological modulation of cardiomyocytes include: (1) Optionally
coat E-PLATES with either fibronectin or other matrix proteins. (2)
Seed either embryonic stem cells (ES cells) of mammalian origin,
mammalian adult stem cell-derived cardiomyocytes or primary
cardiomyocytes isolated directly from mammalian heart tissue at
specific seeding densities to the wells of the device. (3) Allow
the cells to attach and spread. (4) Monitor cardiomyocyte viability
over time using the impedance-monitoring system at prespecified
intervals of time for 12, 24, 48, 72 hours or longer (5) At certain
time after cell seeding, treat the cell with agents that may cause
morphology changes at one or more concentration; using the vehicle
that the agent is dissolved in as a control. (6) Continue
monitoring the cardiomyocytes at 1 minute intervals for at least
1-2 hours to capture any immediate morphological changes and
continue to monitor at 30 minutes intervals of time for additional
12, 24, 48, 72 hours or longer to detect long term morphological
changes (7) Quantify the extent of morphological change by
normalizing the cell index values immediately prior to agent
addition and determine the normalized cell index at a given time
point after agent addition; The extent of morphological change can
be expressed as EC-50 value which quantifies the activity of the
agent with respect to the cardiomyocyte shape changes.
As an example, we describe here the use of the ACEA RT-CES system
to measure and monitor the attachment and growth of mouse ES cells
derived cardiomyocytes and subsequently treated with isoproteranol,
a .beta.2 adrenergic receptor agonist known to induce hypertrophy
(FIG. 3A). Mouse ES-derived cardiomyocytes were seeded at a density
of 25,000 cells per well in ACEA E-PLATES that had been precoated
with fibronectin. The attachment and growth of the cells were
monitored on RT-CES system for 72 hours and then treated with
increasing doses of the compound isoproteranol. According to FIG.
3A, isoproteranol causes a concentration dependent change in cell
index readings. The timing of the cell index change is consistent
with a change in the morphology of the cells which we have shown
previously for other GPCR agonists in primary cells (Yu et al
(2006) Real-time monitoring of morphological changes in living
cells by electronic cell sensor arrays: an approach to study G
protein-coupled receptors; Analytical Chemistry, Vol 78, pages
35-43). To quantify the extent of isoproteranol-induced
morphological changes in mouse ES-derived cardiomyocytes, the
normalized cell indices were plotted against the log of the
corresponding isoproteranol concentrations (FIG. 3B). From the
sigmoidal curve generated a half maximal activity or IC-50 value of
3.1 nM was derived for the compound.
E. Method to Assess and Quantify Cardiomyocyte Beating In Vitro
Using Cell Sensor Impedance Technology
Isolated primary cardiomyocytes as well as ES-derived
cardiomyocytes retain the ability to beat in culture. These cells
provide an excellent model system to study cardiomyocyte function
in vitro, especially with regards to cardiotoxicity. A number of
cardiotoxic drugs are known to affect certain heart channels, such
as the ERG channels, that are involved in excitation-contraction
coupling of cardiomyocytes. Cardiomyocytes have an innate ability
to undergo mechanotransduction, that is that the spontaneous force
generation of the beating cardiomyocyte is translated to
intracellular biochemical signals. Membrane receptors such as
integrins, ion channels and other proteins have been shown to play
a crucial role in cardiac mechanotransduction and lead to a
continuous and rhythmic dynamics of the cardiac actin cytoskeleton
and morphology. Because the impedance-based technology and system
can sensitively and precisely detect transient changes in
morphology and adhesive capacity of the cells, it can be used to
monitor cardiomyocyte beating in vitro.
The steps involved in using an impedance-measurement system for
measurement of cardiomyocyte beating include: (1) Provide a
single-well or multi-well device that comprise microelectrode
arrays in well(s) of the device, which can be used for monitoring
cell-substrate impedance. (2) Optionally coat wells of the device
with either fibronectin or other matrix proteins. (3) Seed either
embryonic stem cells (ES cells) of mammalian origin or primary
cardiomyocytes at specific seeding densities to the wells of the
device. (4) Allow the cells to attach and spread. (5) After a
specified period of time unique to ES-derived cardiomyocytes or
primary cardiomyocytes, monitor cardiomyocyte beating using the
impedance-monitoring system to monitor electrode impedance by using
milli-second kinetic readout to resolve the individual beat cycles
of the cells.
The milli-second kinetic readout requires that the impedance
measurement system can provide impedance measurement data at
milli-second time resolution. In other words, the time difference
between two consecutive impedance measurement for a well shall be
in the range of milli-seconds (e.g., less than 500 milli-second,
less than 300 milli-second, less than 100 milli-second, less than
40 milli-second, less than 30 milli-second, less than 20
milli-second, less than 10 millisecond, or less than 1 millisecond
or faster). The milli-second kinetic readout is required to resolve
the individual beat cycles of the cells. Thus, the time resolution
for the impedance measurement should allow the system to perform
measurement at least two time points for each beat cycle, or at
more than two points for each beat cycle. These milli-second
kinetic readouts may be performed within extracellular recording
experiments by switching between an extracellular recording mode
and impedance mode via a switching means. The fast kinetic readouts
associated with such an impedance based system permits the user to
obtain multiple impedance measurements to obtain the impedance data
for one or more beating cycle of the cardiomyocytes between
extracellular recording measurements.
One example of the impedance measurement systems is an improved
fast-impedance-measurement system from ACEA Biosciences, where the
device is the E-PLATE in the form of microliter plates whose wells
comprise microelectrode structures. An important aspect of the
present invention is that the impedance measurement circuitry
together electronic switching circuitry and the associated is
capable of measuring electronic impedances of the one or more wells
at milli-second time resolution, that is to say, the time
difference between two adjacent impedance measurements for each
well is in the range of milli-seconds. This requirement is
important, especially when the system comprises a device having
multiple wells such as 4 wells, 8 wells, 16 wells or 96 wells. For
example, if the system comprises a device having 96 wells, the
system hardware and software should be capable of measuring the
impedances of all the 96 wells with milli-second time resolution
between two adjacent impedance measurement points for each and
every well. Preferably, the time difference between two adjacent
measurement points for each and every given well is less than 500
milliseconds. More preferably, the time difference between two
adjacent measurement points for each and every given well is less
than 300 milliseconds. More preferably, the time difference between
two adjacent measurement points for each and every given well is
less than 200 milliseconds. Still more preferably, the time
difference between two adjacent measurement points for each and
every given well is less than 100 milliseconds. Still more
preferably, the time difference between two adjacent measurement
points for each and every given well is less than 40 milliseconds.
Still more preferably, the time difference between two adjacent
measurement points for each and every given well is less than 20
milliseconds. Still more preferably, the time difference between
two adjacent measurement points for each and every given well is
less than 10 milliseconds.
A number of improvements in the impedance measurement circuitry,
electronic switching circuitry, communication between impedance
measurement circuitry and software can be used to achieve such
milli-second time resolution. One aspect of improvements includes
the use of fast processing electronic chips for analogue-to-digital
conversion, for parallel digital signal processing and data
calculation with field-programmable gate array (FPGA) and for fast
communication between the impedance measurement circuitry and
software. Another aspect of improvements includes the use of
multiple analogue-to-digital (AD) conversion channels so that
analog electronic signals from multiple channels can be converted
to digital signals simultaneously. Such parallel AD conversion is
important, particular for the system having multiple wells, each of
which's measurement time resolution is required to be in the
milli-second resolution. Another very important aspect of
improvements is to replace typical working mode of "measurement of
one-well's impedance at a time" with a mode of "measurement of
multiple-wells' impedance at a time". In "one-well at a time" mode,
when the software issue a command for measuring one well's
impedance, the measurement circuitry would perform the measurement
for one well including signal generation to the well, converting
the voltage signal and the electric current signal for the well to
digital signal, digitally processing the signals to do impedance
calculation and sending the well's impedance data to the computer
over the communication line between the impedance measurement
circuitry and the computer. The system will not perform any
measurement for another well until the completion of the
measurement of this well and until receiving another command for
the measurement of another well. In "multiple-wells at a time"
mode, the software would issue a command for measuring multiple
wells' impedance. The measurement circuitry would simultaneously or
nearly simultaneously perform signal conversion, signal processing
and impedance calculation for multiple wells. The multiple
impedance data for the multiple wells would be sent over the
communication lines to the computer sequentially with one well's
data at time or simultaneously with more than one well's data being
sent at a time. In this "measurement of multiple-wells' impedance
at a time" mode, the system may be performing multiple tasks
simultaneously, for example, while one well's impedance data is
being measured and calculated, another well's impedance data may be
communicated and sent over the communication lines to the
computer.
The steps involved in using the system for measurement of
cardiomyocyte function may include: (1) Optionally coat E-PLATES
with either fibronectin or other matrix proteins. (2) Seed either
ES cells of mammalian origin or primary cardiomyocytes at specific
seeding densities. (3) Allow the cells to attach and spread. (4)
After a specified period of time unique to ES-derived
cardiomyocytes or primary cardiomyocytes, monitor cardiomyocyte
beating using the RT-CES system by using milli-second kinetic
readout to resolve the individual beat cycles of the cells.
As an example, we describe here the use of the ACEA RT-CES system
to measure and monitor the beating of cardiomyocytes using fast
kinetic software. Mouse ES cells were seeded at a density of
between 3,000 to 50,000 cells per well in ACEA E-PLATES that had
been precoated with fibronectin. The attachment and growth of the
cells were monitored on RT-CES system. FIG. 4 shows the cell index
curves measured on RT-CES system for 4 individual wells, with cell
culture medium as background starting from cell seeding to about 86
hrs after cell seeding. For such long term measurement, cell
electrode impedance and corresponding cell indices were measured at
about 15 minute intervals. As evidenced on these plots, the cell
index curves were rather smooth up to about 44-48 hrs, after which
there were "noises" or "small-spikes" on the cell index curves.
Such spikes were most evident after about 60 hrs. Such spikes in
the impedance or cell index readout are associated with the beating
of the cells. During the synchronized beating of the cells, the
cell morphology and cell adhesion/attachment to the electrodes
change regularly in synchrony with the cell beating. Such regular
or periodic changes in cell morphology and cell adhesion/attachment
are then reflected in the changes in cell-electrode or
cell-substrate impedances.
In order to monitor beating of cardiomyocytes, a specially designed
software and measurement circuit hardware that are capable of
millisecond impedance data acquisition (e.g., typically with time
resolution between consecutive impedance measurement for a same
well being less than 40 milli-seconds) was used to monitor the
quick rhythmic beating of the cardiomyocytes. For such measurement,
the background electrode impedance is measured with the cells
inside the wells (note, this is in contrast with FIGS. 1 and 4,
where the background measurement is performed using cell culture
media). The software, together with specially designed hardware
circuits was used to measure cardiomyocyte beating at distinct
stages throughout its attachment and growth phases (FIG. 5). For a
baseline reference, the impedance measurement was done on the ES
cells at 24 hours where the cells had not fully spread and formed a
tight monolayer and even though the cells appear to beat when
visualized under a microscope, they do so asynchronously and as a
result no net beating signal is detected (FIG. 5A). For plot,
impedance readout has been converted into dimensionless cell
indices. FIGS. 5B and 5C shows that ES cells that had fully spread
and formed tight junctions with neighboring cells at 48 hours and
72 hours respectively, show regular impedance-spikes which
correlate with the beating frequency of cardiomyocytes as judged by
microscopic observation.
To use the measured cell index curves, it is important to further
derive various physiologically relevant parameters. Several
important parameters may include, the beating rate of the
cardiomyocytes (i.e., how many times the cells beat within a unit
of time for example, a minute), the beating amplitude (i.e. the
magnitude of the beating of cells in terms of impedance change) the
average amplitude intensity in a unit time as well as the average
length of time between the beats, time of rise for a beat, time of
decay for a beat. Because of the unique and complex nature of the
impedance readout signals (smaller signal amplitude, sampling time
resolution may be limited by the hardware and the software used),
appropriate methods or techniques are required for analyzing cell
index curves to derive the above mentioned parameters.
For deriving the beating rates of cardiomyocytes, one method may be
by counting how many peaks there are within a given time frame (for
example, one minute). For this approach to work, the sampling time
resolution has to be sufficiently high so that the each beat of the
cells does show a peak on the recorded cell index curves. In
addition, determining a peak "automatically" also require some
algorithm. For example, each peak would have to have one "rise" in
cell index and also one "decay" in cell decay. Each
"rise"-and-"decay" pair forms a single peak. The algorithm needs to
determine such "rise" and "decay" portions of the curves and then
counts a peak.
Another method to derive or count the beating rates of
cardiomyocytes is to perform a detailed signal analysis to derive
the frequency components of the cell index curves and to derive the
magnitude of each frequency components. One method of signal
analysis is Fourier transform of the cell index curve (of the time
domain). Like above method, the sampling time-resolution needs to
be sufficiently high so that each beat of the cells has at least
three time points being measured. After performing Fourier
transform, we would look for the frequency components having the
largest magnitude and such frequency would correspond to, or be
very close to, the beating frequency. In addition, for such
analysis, giving a fixed sampling time resolution, the more time
points sampled for analysis, the more accurate it is for the
analyzed beating frequency. FIG. 6A and FIG. 6B shows a pair of
cell index curves and the corresponding beat rate based the above
described Fourier transform. In FIG. 6A, the cell index curves last
from time zero to time 42 seconds. In FIG. 6B, the beating
frequency for the traces in FIG. 6A is analyzed using the method
described here, i.e., Fourier transform followed by picking up the
highest-magnitude frequency component. For each derived frequency
data at one time moment in FIG. 6B, cell index data from multiple
time points (starting from previous 98 time points plus the time
moment of interest) is used for analysis. Thus, the time axis in
FIG. 6B starts from about 20 seconds to 42 seconds.
Another method deriving the beating rates of cardiomyocytes is to
first determine the time length (.DELTA.T in seconds) between two
consecutive peaks and then calculate the beating rate according to
the formula of "beats per minute=60/.DELTA.T". Thus for each
two-consecutive peaks, one can calculate one beating rate.
Furthermore, one can plot this beating rate as a function of the
time (of the first of the two consecutive peaks) to obtain the time
dependency of the beating rates. FIG. 7A shows an example of the
time-dependent beat rates derived using this method, for mouse
ES-derived cardiomyocytes treated with compound sotalol at a
concentration of 4.4 uM. Corresponding cell index data is shown in
FIGS. 7C and 7D, where the time resolution between two adjacent
points is 40 milli-seconds. In other words, a second in FIGS. 7C
and 7D is equivalent to 40-milli-second.
For deriving the amplitude of the beating of the cardiomyocytes,
there may also be different methods. One method is to analyze each
peak and finding the peak maximum and the peak minimum. The
amplitude is calculated by subtracting peak maximum by the peak
minimum. Then, one can plot the peak amplitude as a function of the
time of the peak to obtain the time dependency of the peak
amplitude. FIG. 7B shows an example of the time-dependent peak
amplitudes derived using this method, for mouse ES-derived
cardiomyocytes treated with compound sotalol at a concentration of
4.4 uM. Another approach may also be to use Fourier transform
described above. Then based on derived Fourier coefficients, one
can re-simulate time domain cell index curves and look for the peak
magnitude from the simulated curves
For deriving the averaged length of time between the beats, there
may also be different methods. For each identified peak, one can
first determine a starting point of the peak. Then the time
difference between two consecutive peaks at the two starting points
of the peak can be used for the length of time between the
beats.
With the method of determining each peak, one can also calculate
the time-of-rise of the peak and the time-decay-of the peak.
F. Method to Assess the Effect of Known Pharmacological Agents,
Proteins, Peptides, Antibodies which Modulate Cardiac Beating Using
the Impedance-Measurement System
The method described in section E above offers a convenient
label-free, real-time and quantitative method for screening of
modulators of cardiac function using a fast impedance-measurement
system capable of milli-second measurement time resolution. The
steps involved in screening for modulators of cardiomyocyte beating
using an impedance-measurement system in vitro include: (1) Provide
a single-well or multi-well device that comprise microelectrode
arrays in well(s) of the device, which can be used for monitoring
cell-substrate impedance. (2) Optionally coat wells of the device
with either fibronectin or other matrix proteins. (3) Seed either
embryonic stem cells (ES cells) of mammalian origin, mammalian
adult stem cell-derived cardiomyocytes or primary cardiomyocytes at
specific seeding densities to the wells of the device. (4) Allow
the cells to attach and spread. (5) After a specified period of
time unique to ES-derived cardiomyocytes or primary cardiomyocytes,
monitor cardiomyocyte beating using the impedance-monitoring system
to monitor electrode impedance by using milli-second kinetic
readout to resolve the individual beat cycles of the cells. This
step should be done immediately prior to addition of
pharmacological agent, in order to obtain a baseline of the
cardiomyocyte beating frequency using fast measurement software and
hardware to ensure milli-second kinetic readout signals. (6)
Addition of the pharmacological agents at one or more doses and
continue monitoring the cardiomyocyte beating frequency.
Similar to Section E, the milli-second kinetic readout requires
that the impedance measurement system can provide impedance
measurement data at milli-second time resolution. In other words,
the time difference between two consecutive impedance measurement
for a well shall be in the range of milli-seconds (e.g., less than
500 milli-second, less than 300 milli-second, less than 100
milli-second, less than 10 millisecond, or less than 1 millisecond
or faster). The milli-second kinetic readout is required to resolve
the individual beat cycles of the cells. Thus, the time resolution
for the impedance measurement should allow the system to perform
measurement at least two time points for each beat cycle, or at
more than two points for each beat cycle.
One example of the impedance measurement systems is a fast
impedance-measurement system from ACEA Biosciences, where the
device is ACEA E-PLATE in the form of microtiter plates whose wells
comprise microelectrode structures. The steps involved in screening
for modulators of cardiomyocyte beating using the fast
impedance-measurement system in vitro include: (1) Seeding
cardiomyocytes, in E-Plates exactly as described in steps 1-4 in
section E above for using the system for measurement of
cardiomyocytes beating. (2) Prior to addition of pharmacological
agent, obtaining a baseline of the cardiomyocyte beating frequency
using the fast kinetic RT-CES hardware and software. (3) Addition
of the pharmacological agents at one or more doses and continue
monitoring the cardiomyocyte beating frequency.
In order to demonstrate the utility of the millisecond kinetic
measurements, we first used two pharmacological agents, one known
to suppress the heart rate and consequently cardiomyocyte beating
and the other known to increase the heart rate and consequently the
rate of cardiomyocyte beating. As Mouse ES cells were seeded in
FN-coated E-PLATES and monitored for about 72 hours when the cells
differentiated into beating cardiomyocytes, as described in Section
E. A baseline of the cardiomyocyte was taken for approximately 40
seconds using the specially designed fast kinetic data acquisition
hardware and software which is capable of millisecond data
acquisition and display. An agonist of muscarinic receptors,
carbachol, which is known to slow down the heart rate was added to
one well at a final concentration of 333 nM and cardiomyocyte
beating was monitored for 10 minutes (FIG. 8A). The data clearly
shows that carbachol significantly slows down the rate of
cardiomyocyte beating from .about.80 beats/min prior to carbachol
addition to .about.60 beats/min after carbachol addition (Table I).
Alternatively, addition of isoproteranol at a final concentration
of 4.4 uM significantly increased the rate of cardiomyocyte beating
from .about.65 beats/min to 115-135 beats/min (FIG. 8B and Table
I). These data clearly show that the readout system and the fast
kinetic software are sufficiently robust and sensitive to detect
these changes in rate of cardiomyocyte beating even at very low
compound concentrations. Similar to the cell index plot shown in
FIGS. 7C and 7D, the time resolution between two adjacent points in
FIGS. 8A and 8B is 40 milli-seconds. In other words, a second in
FIGS. 8A and 8B is equivalent to 40-milli-second.
TABLE-US-00001 TABLE I Beat Pattern Compound Mechanism
Concentration Rate Amplitude Change Carbachol Muscarnic receptor
333 nM From From 0.07 Beating rate agonist ~80 to to 0.06 decreased
~60 Isoproteranol b2 Adrenergic 4.4 uM From From 0.19 Beating rate
receptor agonist ~65 to to 0.16 increased 115-136
To further demonstrate the capabilities of the impedance-based
monitoring of cardiomyocyte beating in detecting drugs which may
adversely affect heart function, a number of drugs which have been
pulled out of the market due to cardiotoxic side effects such as
ERG channel inhibition and QT elongation were compiled and tested
in a dose-dependent manner. The list of these compounds, their
mechanism and adverse side affects are shown in Table II. For these
tests, mouse ES derived cardiomyocytes were seeded at a final
density of 25,000 cells in ACEA E-PLATES and continually monitored
by the improved impedance-measurement system. Approximately, 72
hours after cell seeding the fast impedance-measurement system
including fast kinetic software and fast measurement hardware
circuitry was used to establish a baseline reading of cardiomyocyte
beating for each well for about 40 seconds. Subsequently, the cells
in each well were treated with the indicated drug and dose shown in
FIG. 9. Similar to the cell index plot shown in FIGS. 7C and 7D,
the time resolution between two adjacent points in all the figures
in FIG. 9 is 40 milli-seconds. In other words, a second in a figure
in FIG. 9 is equivalent to 40-milli-second.
Table II shows a summary of the results, clearly demonstrating that
compounds which have been shown to affect ERG channels do affect
various aspects of cardiomyocyte beating and function such as
frequency of beating, magnitude of beating. Furthermore, as shown
in FIG. 10 some of these compounds can lead to qualitatively
different or similar patterns. For example, the compounds E4031,
Astezimole and dofetilide which are ERG channel inhibitors do
contain patterns with similar features. In summary these results
clearly indicate that using the fast kinetic software along with
the RT-CES system do lead to a sensitive and robust readout for
cardiomyocyte beating that can also detect drugs which are known to
be cardiotoxic. Similar to the cell index plot shown in FIGS. 7C
and 7D, the time resolution between two adjacent points in all the
figures in FIG. 10 is 40 milli-seconds. In other words, a second in
a figure in FIG. 10 is equivalent to 40-milli-second.
TABLE-US-00002 TABLE II Compound Mechanism Concentration Beat Rate
Amplitude Pattern Change Astemizole anti-histamine 400 nM 0
<0.01 From 68-72 bpm to irregular being to beating stopped
Terfenadine anti-histamine 200 nM 0 <0.01 From 71-78 bpm to
irregular beating to beating stopped Erythromycin anti-biotic 13.3
uM From ~60 to 0.09 to 0.07 No pattern change ~80 Moxifloxacin
anti-biotic 20 uM From ~80 to From 0.055 to No pattern change ~73
0.060 Pentamidine anti-infective 20 uM From 71~78 From 0.24 to No
pattern change to ~71 0.21 Amitriptyline Serotonergic 4.4 uM From
~70 to From 0.22 to No pattern change Inhibitor ~90 0.17 Verpamil
Ca channel 130 nM From ~65 to 0 From 0.13 to From ~65 bpm to
blocker 0.06 only occasional single beating. Rosglitazone PPAR
agonist 13.3 uM From ~79 to From 0.22 to No pattern change ~75 0.17
Dofitlite 500 nM From ~80 to From 0.09 to Pattern changed, ~180
0.02 much faster Rofecoxib COX-2 13.3 uM From ~68 to From 0.2 to No
pattern change Inhibitor ~60 0.19 Rofecoxib COX-2 40 uM From ~62 to
From 0.16 to Pattern change, no Inhibitor ~60 0.10 beating after
initial treatment, then recovers Celecoxib COX-2 4.4 uM From ~60 to
From ~0.2 to Pattern change Inhibitor (~20-~50) ~0.12 Doxirubicin
Anthracycline 40 uM ~70 ~0.16 No pattern change (initially).
Beating pattern changes after 2 hrs. Cyclosporin A Calcineurin 13.3
uM From ~70 to From 0.18 to No pattern change inhibitor ~80 0.15
Propalanol .beta.2-adrenergic 4.4 uM From ~70 to From 0.25 to
Pattern changed, receptor over 150 0.025 much faster and antagonist
irregular Sotalol 13.3 uM From ~80 to From 0.27 to Much faster,
~160 0.07 pattern changed E4031 K channel 120 nM From 80 to From
0.09 to Pattern changed, inhibitor 160 0.03 much faster DDT
Pesticide 8 uM From 80 to From 0.25 to Initially become 140, then
to 0.02 faster, later ~80 irregular beating PCB Organic 8 uM From
~65 to o From 0.2 to 0 Beating stopped toxicant Endosulfan
insecticide 8 uM From ~75 to o From 0.2 to 0 Beating stopped
To further demonstrate the capabilities of the impedance-based
monitoring of cardiomyocyte beating in detecting drugs which may
adversely affect heart function, compounds at different
concentrations were tested to demonstrate the dose-dependent
effects of these compounds on cardiomyocytes. Approximately, 72
hours after cell seeding the impedance-measurement system including
software and hardware capable of milli-second measurement time
resolution was used to establish a baseline reading of
cardiomyocyte beating for each well for about 40 seconds.
Subsequently, the cells in each well were treated with drugs at
different dose concentrations. FIG. 11 shows an example of dose
dependent effects of Astemizole on cardiomyocytes beating at
different concentrations. At high concentration of 400 nM,
Astemizole had such a strong effect on the beating of
cardiomyocytes that the beating almost stopped. The effect of
Astemizole on the beating of the cardiomyocytes is clearly
does-dependent. At low concentration of 40 pM, its effect on the
beating of the cardiomyocytes is small that the cardiomyocyte
beating rate was not affected. Similar to the cell index plot shown
in FIGS. 7C and 7D, the time resolution between two adjacent points
in all the figures in FIG. 11 is 40 milli-seconds. In other words,
a second in a figure in FIG. 11 is equivalent to
40-milli-second.
G. Method to Assess the Developmental and Functional Consequence of
Specific Gene Knockout and Transgene Expression in Es-Derived
Cardiomyocytes
The ES cells offer a suitable experimental model system that is
amenable to genetic manipulation. Therefore, specific genes can be
targeted in knockout experiment as well as genes can be expressed
in a developmental or stage specific manner under the control of
special promoters. The impedance-based measurement system can be
used to evaluate the role of these genes in cardiac viability,
morphology, development and or beating function. The steps involved
in assessing the developmental and functional effect of gene
knockout or transgene expression include (1) Obtain ES cells
harboring specific knockout of genes or which expresses a
particular transgene. (2) Provide a single-well or multi-well
device that comprise microelectrode arrays in well(s) of the
device, which can be used for monitoring cell-substrate impedance.
(3) Optionally coat wells of the device with either fibronectin or
other matrix proteins. (4) Seed the embryonic stem cells (ES cells)
of mammalian origin or adult stem cells of mammalian origin at
specific seeding densities to the wells of the device. (5) Allow
the cells to attach and spread and monitor the growth and viability
of the cells using an impedance measurement system. (6) After a
specified period of time unique to ES-derived cardiomyocytes or
primary cardiomyocytes, monitor cardiomyocyte beating using the
impedance-monitoring system to monitor electrode impedance by using
milli-second kinetic readout to resolve the individual beat cycles
of the cells. (7) If a particular gene is required for development
of cardiomyocytes from ES cells, it is likely that the knockout of
that gene will either affect the viability of the cells or block or
delay the differentiation of ES cells to cardiomyocytes. Since the
impedance-based measurement system is capable of functional
monitoring of cardiomyocyte both in long term assays and short term
assays, it can be used as a specific way to monitor the effect of
either gene knockout or transgene expression on cardiomyocyte
function. (8) Alternatively, the ES cells can be transfected with
specific siRNA to "knockdown" the product of a particular
transcript and then monitor cardiomyocyte viability,
differentiation and function in vitro using the impedance-based
measurement system.
Similar to Section E, the milli-second kinetic readout requires
that the impedance measurement system can provide impedance
measurement data at milli-second time resolution. In other words,
the time difference between two consecutive impedance measurement
for a well shall be in the range of milli-seconds (e.g., less than
500 milli-second, less than 300 milli-second, less than 100
milli-second, less than 10 millisecond, or less than 1 millisecond
or faster). The milli-second kinetic readout is required to resolve
the individual beat cycles of the cells. Thus, the time resolution
for the impedance measurement should allow the system to perform
measurement at least two time points for each beat cycle, or at
more than two points for each beat cycle.
One example of the impedance measurement systems is an improved,
fast impedance-measurement system from ACEA Biosciences, where the
device is ACEA E-PLATE in the form of microtiter plates whose wells
comprise microelectrode structures. The steps involved in assessing
the developmental and functional effect of gene knockout or
transgene expression using the fast impedance-measurement system in
vitro include: (1) Obtain ES cells harboring specific knockout of
genes or which expresses a particular transgene. (2) Follow steps
1-4 of Section D for using the fast impedance-measurement system
for measurement of cardiomyocytes function. (3) If a particular
gene is required for development of cardiomyocytes from ES cells,
it is likely that the knockout of that gene will either block or
delay the differentiation of ES cells to cardiomyocytes. Since the
system is capable of functional monitoring of cardiomyocyte, it can
be used as a specific way to monitor the effect of either gene
knockout or transgene expression on cardiomyocyte function. (4)
Alternatively, the ES cells can be transfected with specific siRNA
to "knockdown" the product of a particular transcript and then
monitor cardiomyocyte differentiation and function in vitro using
the fast impedance-measurement system.
H. Devices and Method for Extracellular Recording
Devices and systems of the present invention permit extracellular
recording of cell populations using a variety of technical
approaches. Extracellular recording may be performed alone or
together with impedance monitoring of cells. As discussed in prior
sections, while the present device is suitable for performing
extracellular recording of various cells and/or tissues, the
present invention is particularly useful for the extracellular
recording of cardiomyocyte cells, cardiomyocyte precursor cells, as
well as tissues that contains such cells.
The devices and systems of the present invention provide structural
configurations that functionally establish cell-free zones to
prevent direct interaction between cells and reference electrodes,
provide enhanced measurement reliability and accuracy, and bridge
the current gap between impedance monitoring and extracellular
recording of cells.
For instance, in a first approach, the devices and systems provide
a cell-free zone for the positioning of a reference electrode.
Accordingly, while traditional systems often require stringent
physical parameters for the reference electrode for enhanced
measurement, such as stringent tolerances related to surface area,
resistance, impedance and the like, present invention provides an
alternative solution. Specifically, in one embodiment, a device for
performing extracellular recording of cells, such as
cardiomyocytes, is provided which includes a nonconductive
substrate forming or provided as a base of one or more wells; an
extracellular recording electrode positioned on the substrate
within the well, wherein the recording electrode is accessible to
cells when a cell sample is added to the device; and a reference
electrode positioned within the well in a cell-free zone, the
cell-free zone is characterized as free from contact with cells
when the cell sample is added to the device, thereby preventing
contact between cells and the reference electrode. That is, the
reference electrode is a cell-free electrode during operation.
Extracellular recording is conducted by amplifying and recording
electrical voltage signals between the recording electrodes and
reference electrodes. Such electrical voltages are induced on the
electrodes as a result of ionic current or movement through cell
culture media or solution supporting the cells during the
experiment as a result of opening and/or closing of different ion
channels across cell membrane during the action potential duration.
Generally, it is desirable and it is recognized for the reference
electrodes to have small electrode impedances. The small electrode
impedance is achieved by using reference electrodes with large
effective surface areas by increasing the ratio of the surface area
of the reference electrodes to that of recording electrode by a
factor of a hundred, even thousands of times. FIG. 15 shows a
schematic representation of such electrode pairs placed on a
non-conductive substrate, including a small area recording
electrode 1501 and a large area reference electrode 1502. For
clarity, the connection pads on the substrate for connecting the
recording electrode and reference electrode to external circuitry
are not shown in FIG. 15. For example, in previous extracellular
recording devices, the recording electrodes and reference
electrodes are all incorporated onto a substrate surface within a
fluid-container well where the recording electrodes are of simple
electrode geometry (e.g. circle, or square) having a diameter of
10-100 microns in diameter whereas the reference electrodes would
have much larger surface areas.
For the present invention, it is recognized that the extracellular
recorded voltage signals are recorded as the difference in the
electrical potentials between the recording electrode and reference
electrode. In order to achieve improved consistency and
reproducibility of the recorded voltage signals, it is desirable to
minimize the contribution of any electrical signal from the
reference electrode to the recorded voltage signals and to ensure
that the majority, if not all, of the recording voltage signals are
derived from that on the recording electrode. As mentioned above,
the previous extracellular recording devices are designed to this
goal by reducing the electrode impedance of the reference electrode
through using reference electrode of a large surface area and so
minimizing the voltage signals on the reference electrode.
In one aspect, the present invention is directed to a device for
extracellular recording of cells, the device comprising: a
nonconductive substrate forming a base of one or more wells; a
recording electrode positioned on the substrate within the well,
wherein the recording electrode is accessible to cells when a cell
sample is added to the device; and a reference electrode positioned
within the well in a cell-free zone, the cell-free zone
characterized as free from contact with cells when the cell sample
is added to the device, thereby preventing contact between cells
and the reference electrode. In one embodiment, the recording
electrode has a diameter from about 10 um to about 200 um. In
another embodiment, the recording electrode comprises an electrode
structure comprising a plurality of electrode elements. In
preferred embodiments, the reference electrode is positioned on the
substrate in the cell-free zone.
Now with reference to particular configurations, the device for
extracellular recording of the cells is designed and constructed so
that the reference electrode is positioned in the well in a
cell-free zone so that when the cells are added to the device, the
cells would not be in contact with the reference electrode. In
comparison with the situation where the cells are in contact with
the reference electrode, no-cell-contact with the reference
electrode would significantly reduce the contribution of the
electrical signals from the reference electrode so that the
amplified and recorded voltage signals would mainly come from the
recording electrode. Consider the following. When the cells are in
contact with the reference electrodes, the ionic current and ionic
movement in the regions close to the cells due to the opening
and/or closing of ion channels (and/or other ion transporters) in
the cell membrane would directly cause an electrical potential
signal on the close-by reference electrode since these reference
electrodes are in direct contact with the cells. On the other hand,
when there is no cell in direct contact with the reference
electrode, only the ionic current and ionic movement due to the
opening and/or closing of the ion channels in the membranes of
other cells may result in small, if any, electrical potential on
the reference electrode since these cells are not in contact with
the reference electrode. The induced electrical potential on the
reference electrode due to such no-contact-cells located at other
regions of the wells would be significantly smaller that that
induced by the cells in direct contact with reference electrode,
because the ionic movement/current in the media would be much
smaller in the reference electrode region which is away from the
cells.
There are different approaches to position the reference electrode
within the well in a cell-free zone to prevent contact between
cells and the reference electrodes. In one embodiment, the
reference electrode is positioned on the substrate forming or
provided as the base of the wells to which the cells are added. The
reference electrode is located in a region that is separated by a
barrier from the region containing the recording electrode.
Preferably, for the device of the present invention, the reference
electrode may be separated by a barrier from the recording
electrode so that when the cells are added to the wells containing
the recording electrode, the cells will be prevented from landing
on and contacting with the reference electrode. When the cells are
added to the recording electrode region, the cells are prevented
from moving to the reference electrode region and prevented from
contacting the reference electrode. Example of this embodiment is
shown in FIG. 33 where both top view and cross-sectional view of a
device of the present invention are shown. Here, the recording
electrode 3301 and reference electrode 3302 are incorporated on the
substrate 3304. The barrier 3303 is placed inside the well 3305,
separating the recording electrode and reference electrode. In use,
the cell culture media (or appropriate solutions for supporting the
cells during the experiment) containing the cells is added first to
the left half of the well 3305 to the recording electrode region,
ensuring that the solution would not overflow above the barrier to
the reference electrode. After all the cells settle down to the
substrate (which may take some minutes to typically less than an
hour), the culture media (or appropriate solutions for supporting
the cells during the experiment) not-containing the cells can be
added to the right half of the well and sufficient media (or
appropriate solutions for supporting the cells during the
experiment) is added so that the media would overflow above the
barrier, the final media height in the well would be above the
barrier and there would be electrical connection path between the
recording electrode and reference electrode through the media. Note
that for simplicity of the figure, the electrical connection traces
for the reference electrode and the recording electrode to the
connection pads and the connection pads on the substrate are not
shown in FIG. 33. In a related embodiment, the barrier could be
provided in a configuration having a series of permeable apertures
that selectively permit flow of media while selectively preventing
the migration or movement of cells into the cell-free zone;
however, such a configuration may lend to clogging of the apertures
by blocking cells. In an exemplary embodiment of this approach, the
reference electrode may be located under the permeable aperture
structure so that the media would flow to the reference electrodes
whilst the cells would not.
In another embodiment, the barrier is in a form of a plug that is
made of biocompatible materials and can be placed directly above
and on the reference electrodes. When the cells are added to the
well, the cells are prevented by such a plug to be in contact with
the reference electrode and the cells would land on other regions
of the substrate, including the recording electrode region. After
the cells settle down and adhere to the substrate of the well, the
plug is removed and the media or solution in the well would move
into the reference electrode region. A complete circuit is formed
between the recording electrode and the reference electrode through
the media or solution in the well.
Barriers may take alternative forms. For example, barriers may
include ledges, walls, troughs and the like to act as a structural
or physical barrier to cells. In some instances, a cell repulsive
gel is applied to the substrate to define the cell-free zone. The
barrier should however permit the medium to contact the reference
electrode such that the recording and reference electrode are
operably linked through a conductive media. Barriers may be
permanently fixed to the device or may be removable as the
configuration permits. Similarly, the bather may be formed from any
suitable material and have any suitable dimensions consistent with
isolating the reference electrode from cells, while obtaining
extracellular recording measurements. In some embodiments the
barrier is constructed from the same material as the nonconductive
substrate. In another embodiment, the reference electrode is
positioned on a different plane from the recording electrode,
ensuring that the cells added to the recording electrode region
would not be in contact with the reference electrode. In such an
embodiment the cell-free zone may be a raised surface to prevent
cell migration or spreading onto the cell-free zone. In other
embodiments an angled surface prevents migration upward along the
angled surface and thus provides a suitable cell-free zone towards
the top of a slope. In another embodiment, the cell-free zone is a
region encapsulated by a porous covering, which permits passage of
medium while preventing access by cells.
In still another embodiment, the reference electrode is an external
electrode free from contact with the substrate to which the
recording electrode is positioned. For instance, a cell-free zone
may be spatially positioned remote from the substrate but within a
volume of the well. The schematic representation of such a device
is shown in FIG. 34. A recording electrode 3401 is positioned on
the substrate 3404. The reference electrode is an external wire
electrode (e.g. gold wire, or Silver-silver chloride wire electrode
if they are placed into the well for very short time, not causing
cytotoxic effects) 3402. In FIG. 34, an impedance electrode array
3406 is also positioned on the substrate 3404. The electrode array
3406 is of straight-line, interdigitated electrode geometry and
comprises two electrode structures, each of which comprise multiple
electrode elements. In use, when the cells are added to the well
3405 containing the recording electrode 3401 and impedance
measurement electrode array 3406, the external reference electrode
3402 can be outside of the well. After the cells are added to the
wells and are settled down to the bottom, the external reference
electrode 3402 can be added to the well manually or automatically
and the reference electrode would not be in contact with the
substrate of the well and not be in contact with the cells on the
substrate. In some embodiments, the external reference electrode
3402 is fabricated to a lid or cover which may be placed over top
of the well.
Thus, a cell-free zone may be spatially positioned away from the
substrate. In such a configuration the cell-free zone may occupy a
volume of the well that does not contact the substrate, yet does
contact medium when added to the device. Accordingly, while cells
do not contact the cell free zone, an electrically conductive media
may still ensure operable contact between an extracellular
recording electrode and a reference electrode.
For extracellular recording, the recording electrode is generally
of simple geometry and consists of a single electrode element such
as a circle, a square or some other geometry. The size of such
recording electrode typically ranges from about 10-30 to about 100
micron in a diameter. Such small electrode geometry has advantages
of recording the electrical potential generated by a small number
of the cells located on the recording electrodes. Action potentials
from such a small number of the cells tend to be synchronized or
nearly synchronized, allowing for a better time resolution for
recording extracellular potential and for resolving different
features of the recorded potential. However, one limitation of such
extra-cellular recording is that due to small area of such
electrodes, there tends to be large variations in recorded signals
between different recorded electrodes of the same geometry in the
same wells (if multiple recording electrodes are positioned inside
a single well) or different wells. In particular, if insufficient
number of the cells is added to the well to cover all the recording
electrodes, it is possible that some recording electrodes may not
show any recorded signal or only show recorded signals of very
small magnitude. Furthermore, depending on exact distribution or
locations of the cells on the recording electrodes, different
recording electrodes may show significantly different extracellular
potential waveforms. For this reason, many existing devices for
extracellular recording comprise multiple small-area recording
electrodes. Extra-cellular potentials from each such recording
electrode are amplified and recorded separately. The user would
pick and choose appropriate signal waveforms recorded for some
individual recording electrodes for data analysis.
To overcome such a limitation, in some embodiments of the present
invention, electrode structures having larger surface areas and
including multiple electrode elements are used as recording
electrodes. The advantage of recording extracellular potential with
such electrode structures is that the extracellular potential is an
integration of the potentials from all the cells on the electrode
array and would be more reproducible between different wells. The
variation in recorded signals between difference electrode
structures from different wells would be smaller than that recorded
with small, single recording electrode elements. For such devices,
each well may comprise a single electrode structure, or two
electrode structures. In preferred embodiment, each well comprises
a single electrode structure consisting of multiple electrode
elements.
Thus, the extracellular recording electrode may be provided in a
variety of configurations. In some instances the recording
electrode includes a unitary, circular or unbranched configuration.
In other embodiments the extracellular recording includes an
electrode structure, which itself may include a plurality of
electrode elements. Exemplary electrode structure and electrode
element configurations may be found in U.S. Pat. Nos. 7,470,533;
4,459,303; 7,192,752; 7,560,269; and 7,468,255, each of which is
incorporated by reference. However, the majority of the above
listed patents refer to arrays of electrodes having at least two
complementary electrode structures. Thus, when considering the
configurations for use as an extracellular recording electrode
structure or element, the skilled artisan would typically provide
one half of the array as set forth in the patents.
FIG. 13 shows more specific examples of such electrode structures
that could be used as recording electrode for recording
extracellular potentials. In FIG. 13A, a single electrode structure
is shown, comprising multiple rectangular electrode elements 1301
that are connected together with an electrode bus 1303. Similarly,
FIG. 13B comprises a single electrode structure, comprising
multiple circle-on-line electrode elements 1311 that are connected
together with an electrode bus 1313. For FIG. 13C, two electrode
structures are shown, one electrode structure comprising multiple
electrode elements 1331 and having electrode bus 1333, and the
other electrode structure comprising electrode elements 1332 and
having electrode bus 1335. The extracellular recording can be
achieved using either electrode structure or both electrode
structures connected together. FIGS. 22 and 23 show example of
recording extracellular potentials using circle-on-line electrode
structures as recording electrode and external wire electrode as
reference electrode. Whilst the recorded waveform is different from
those obtained with small recording electrode, the recorded signals
are very reproducible between different wells.
By way of yet another example, a device for performing
extracellular recordings of excitable cells in vitro, may include,
a) a nonconductive substrate; b) one or more wells on the
substrate; c) one or more electrode structures fabricated on the
substrate, each electrode structure is associated with one of the
one or more wells; and d) one or more reference electrodes external
to the substrate or free from contact with the substrate, each of
which can be inserted into one of the one or more wells, wherein
for each of the one or wells, the corresponding electrode structure
and reference electrode form an electrode pair. In further
embodiments, the substrate has a surface suitable for attachment of
excitable cells, wherein the attachment of excitable cells on the
substrate can result in detectable extracellular recording
potentials between each of electrode pair. FIG. 12 shows another
illustrate example of such electrode pair, comprising a recording
electrode structure on the substrate and a reference electrode
external to the substrate. In a particularly preferred embodiment,
each electrode structure comprises multiple electrode elements. Use
of such a configuration is believed to increase the sampling size
and thus minimize or reduce variation between cell populations. In
another preferred embodiment, each electrode structure comprises
multiple electrode elements, forming half of an interdigitated
electrode array (FIG. 13A). In another preferred embodiment, each
electrode structure comprises multiple electrode elements so that
these electrode elements form a complete interdigitated electrode
array (13C) wherein during extracellular recording the electrode
elements within entire interdigitated electrode array are connected
together. In another preferred embodiment, each electrode structure
comprises multiple electrode elements which have different
electrode element shapes, including rectangular shape, or
sinusoidal shape, spiral or circle-on-line shape. FIG. 13 shows
examples of electrode structures having multiple electrode elements
that can be used for extracellular recording where 13A, 13B, 13C,
13D, 13E and 13F are the rectangular shape, circle-on-line shape, a
complete interdigitated electrode array, sinuosoidal shape,
castellated shapes and a complete circle-on-line electrode array,
respectively. FIG. 14 shows the examples of such electrode
structures for extracellular recording where a recording electrode
structure and a reference electrode are incorporated on the
substrate to form a pair of electrodes. Preferably, the reference
electrode is separated from the recording electrode by a barrier to
the cells so that the reference electrode is in a cell-free zone
when the cells are added to the electrodes. Clearly, these
electrode structures are significantly different from
microelectrode arrays formed by simple electrodes such as
circle-shaped electrodes and/or square-shaped electrodes currently
used in the field of extracellular recording.
In another preferred embodiment of the device for performing
extracellular recording, each electrode structure occupies a
substantial percentage of surface area of the well that the
electrode structure is associated with. In some preferred
embodiments, percentage of surface area of the well at the bottom
being occupied by the electrode structures is more than 5%.
Preferably, percentage of surface area of the well at the bottom
being occupied by the electrode structures is more than 10%. More
preferably, the percentage of surface area of the well at the
bottom being occupied by the electrode structures is more than 20%.
More preferably, the percentage of surface area of the well at the
bottom being occupied by the electrode structures is more than 30%.
More preferably, the percentage of surface area of the well at the
bottom being occupied by the electrode structures is more than 50%.
More preferably, the percentage of surface area of the well at the
bottom being occupied by the electrode structures is more than 70%.
More preferably, the percentage of surface area of the well at the
bottom being occupied by the electrode structures is more than 85%.
Clearly, such large-surface-area electrodes are significantly
different from microelectrode arrays currently used in the field of
extracellular recording, where recording electrodes typically have
a round disc shape or a square shape of smaller size with typical
dimension of 10 to 100 micron in diameter.
In another aspect of the present invention, a method performing
extracellular recording of excitable cells comprises, providing a
device of the present invention, adding excitable cells to the
wells; providing an extra-cellular-recording amplifier that can
measure and record voltage signals at microvolt levels, connecting
electrodes on the devices to the extra-cellular-recording
amplifier; monitoring and recording extracellular potentials at the
electrodes of the devices. Preferably, the method further comprises
analyzing recorded extracellular potential waveforms. More
preferably, the method further comprises adding a compound to the
well, measuring and recording extracellular potentials prior to and
after the compound addition; analyzing extracellular potential
waveforms.
I. Devices and Method for Parallel Extracellular Recording and Cell
Impedance Measurement
In still another aspect of the present invention, a device allowing
for parallel extracellular recording and cell impedance measurement
is provided. Thus, a single device or system is capable of
performing both impedance monitoring of cells and extracellular
recording. Thus, the device may include a means for cell impedance
measuring and a means for extracellular recording operably coupled
to the same substrate. In some instances, impedance monitoring
occurs before extracellular recording measurements. Such an
approach may permit monitoring, determining or confirming desired
cell viability, morphology and the like as set forth in prior
sections prior to initiating extracellular recording assays. Thus,
impedance monitoring permits the user to confirm cellular phenotype
or attributes prior to initiating extracellular recording. In other
applications, the impedance measurement permits the monitoring of
the beating of cardiomyocytes, the monitoring of the change to cell
morphology, cell adhesion, cell viability or other properties after
a compound treatment. Further, impedance monitoring may be
conducted while performing extracellular recording or after
extracellular recording. Impedance monitoring may be used to
monitor cell beating, viability, morphology and the like. While
impedance monitoring and extracellular recording may be performed
over the same time period, the actual measurements may be obtained
at different time points. That is, a switching means may permit the
device to switch from an impedance monitoring mode to an
extracellular recording mode. The combined approach is facilitated
in part due to the increased impedance based time resolution. That
is, impedance monitoring with millisecond time resolution permits
such measurements to be conducted throughout a variety of time
windows, without adversely affecting extracellular recording
measurements.
The skilled artisan will appreciate parallel extracellular
recording may be performed using a variety of configurations
provided herein. In a first example, parallel recording is
performed using a device that includes both a pair of electrodes
for impedance monitoring and a pair of electrodes for extracellular
recording, though pairs of electrodes may include a third or more
electrode. Each pair is selectively operated via a switching means
able to activate either an impedance monitoring mode, which allows
for the connection of the impedance electrodes to external
impedance measurement circuitry; or extracellular recording mode,
which allows for the connection of the extracellular recording
electrode and reference electrode to external voltage amplifier and
signal measurement and recording circuits. In preferred
embodiments, the switch is a programmed switch with adjustable
parameters through a computer interface. Thus, in one example, a
device for parallel impedance monitoring and extracellular
recording of cells is provided, wherein the device includes: a
nonconductive substrate forming or provided as a base of one or
more wells; at least two impedance electrodes capable of monitoring
impedance of the cells, the at least two impedance electrodes
positioned within a well and on the nonconductive substrate,
wherein the at least two impedance electrodes are accessible to
cells when a cell sample is added to the device; an extracellular
recording electrode positioned on the substrate within the well,
wherein the recording electrode is accessible to cells when the
cell sample is added to the device; and a reference electrode
positioned within the well. In the preferred mode, the reference
electrode is positioned in a cell-free zone, which is characterized
as free from contact with cells when the cell sample is added to
the device. When in impedance mode, the pair of impedance
electrodes permits impedance monitoring of cells. When in
extracellular recording mode the extracellular recording electrode
and reference electrode form a pair of electrodes for extracellular
recording measurements. In a related embodiment, a device is
provided that includes a nonconductive substrate, having a surface
suitable for attachment of excitable cells, one or more wells on
the substrate; for each well, a pair of impedance measurement
electrodes, wherein the attachment of excitable cells on the
substrate can result in a detectable impedance change; for each
well, a pair of extra-cellular recording electrodes comprising a
recording electrode and a reference electrode, wherein the
attachment of excitable cells on the substrate can result in a
detectable extra-cellular recording potentials between the
recording-electrode and reference-electrode pair. Preferably, the
pair of impedance measurement electrodes is located on the
substrate. Preferably the extracellular recording electrodes are
located on the substrate and in the same plane as the impedance
electrodes. Also, preferably in such a configuration, the reference
electrode is provided in a cell-free zone. The cell-free zone
prevents direct contact between cells and the reference electrode.
In some instances, a physical barrier prevents contact between
cells and the reference electrode. In other instances, the
cell-free zone is provided on a surface that is not planar with the
impedance monitoring electrodes such that the reference electrode
is free from contact with cells added to the device. In still
further instances, the cell-free zone is spatially positioned
within the well but does not contact the substrate. In such
instances, one recording electrode may be located on the substrate
and another electrode may be located external to the substrate, but
within a volume of the well. Positioning the reference electrode in
the volume of the well free from contact with cells and the
substrate permits electrically conductive medium to complete an
electrical circuit between the recording electrode and reference
electrode; however direct contact between cells and the reference
electrode may be avoided.
In some embodiments, the recording electrode has a diameter from
about 10 um to about 200 um. In other embodiments, the recording
electrode comprises an electrode structure comprising a plurality
of electrode elements
In other configurations, one of the impedance monitoring electrodes
is provided as an extracellular recording electrode. FIG. 16 shows
an example of such device where in one well, a pair of impedance
measurement electrodes is located on the substrate and one of the
impedance measurement electrode structures is used as an
extracellular recording electrode, together with an externally
applied reference electrode. In such a configuration a switching
means may selectively switch between coupling signal between the
two impedance electrodes in an impedance mode and between the
impedance electrode and a reference electrode in an extracellular
recording mode. More particularly, in one embodiment the device for
parallel extracellular recording includes a nonconductive substrate
forming or provided as a base of one or more wells; at least two
impedance electrodes capable of monitoring impedance of the cells,
the at least two impedance electrodes positioned within a well and
on the nonconductive substrate; and a reference electrode, wherein
a first impedance electrode from the at least two impedance
electrodes is electrically coupled to the reference electrode for
performing an extracellular recording measurement at the first
impedance electrode. In such configurations, preferably the at
least two impedance electrodes each have the same surface area and
each comprise an electrode structure comprising a plurality of
electrode elements. While in some embodiments the reference
electrode is positioned on the substrate in other embodiments the
reference electrode is an external electrode free from contact with
the non-conductive substrate.
In still another aspect of the present invention a device for
parallel extracellular recording includes a nonconductive substrate
forming or provided as a base of one or more wells; at least two
impedance electrodes capable of monitoring impedance of the cells,
the at least two impedance electrodes positioned within a well and
on the nonconductive substrate; and an extracellular recording
electrode, wherein a first impedance electrode from the at least
two impedance electrodes is electrically coupled to the
extracellular recording electrode to act as a reference electrode
for performing an extracellular recording measurement. That is, the
impedance electrode may also operate as a reference electrode for
the extracellular recording electrode. In such configurations,
preferably, the at least two impedance electrodes each have the
same surface area and each comprise an electrode structure
comprising a plurality of electrode elements. In one specific
embodiment, the recording electrode for extracellular recording is
in the form of a unitary electrode structure and the reference
electrode is one impedance electrode comprising multiple electrode
elements. For the devices of such configuration, the cells would
contact the impedance electrodes and contact the reference
electrode. This is different from other embodiments of the devices
used for extracellular recording described above. The advantage of
this approach is that the electrode design for parallel impedance
monitoring and extracellular recording is simplified. Instead of
using four electrodes with two electrodes for impedance monitoring
and two electrodes for extracellular recording, three electrodes
are used with one impedance electrode being used for impedance
monitoring and used as a reference electrode for extracellular
recording. For such configuration, the extracellular recorded
voltage signals would have the contributions from extracellular
potential from both recording electrode and the reference
electrode.
The skilled artisan will recognize the device may be operable
linked to a system including an impedance analyzer, extracellular
recording amplifier, electronic switches, electrical noise
filtering circuits, switching means capable of switching
measurement between the at least two impedance electrodes and the
first impedance electrode and the reference electrode and the like.
The skilled artisan will also recognize the system may include
software to instruct the desired measurements or to set desired
parameters for testing or operation.
In still another aspect of the present invention, the method for
parallel measurement of cell-substrate impedance and extra-cellular
potentials comprises a) providing a device allowing for parallel
extracellular recording and cell impedance measurement; b) adding
excitable cells to the wells of the device; c) providing an
impedance analyzer; d) providing an extracellular potential
amplifier; e) connecting electrodes of the devices to the impedance
analyzer; f) connecting electrodes of the devices to the
extra-cellular potential amplifier; g) performing parallel
measurement of cell-substrate impedance and extracellular
potentials. Preferably, the method further comprises adding
compounds to the cells and monitoring cell-substrate impedance and
extracellular potentials prior to and after compound addition.
Preferably, the method further comprises analyzing measured time
dependent impedance responses. Still preferably, the method further
comprising analyzing extracellular-recording wave forms.
J. Devices and Method for Monitoring Cardiomyocyte Beating Using
Label-Free Method
In another aspect of the present invention, a label-free method is
used to monitor cell-substrate interaction for quantifying and
measuring the beating of cardiomyocytes. The label-free method
refers to any method that can be monitor or measure the cells
without the need of using labeling reagents or molecules. Cell
impedance measurement method is one example of label-free
approaches. Another label-free method includes the use of Resonant
Waveguide Grating (RWG) biosensor (see reference, Y. Yang,
Label-Free Cell-Based Assays with Optical Biosensors in Drug
Discovery, in ASSAY and Drug Development Technologies, Volume 4,
Number 5, 2006, 583-595; Y. Yang et al., Label-Free Cell-Based
Assays for GPCR Screening, in Combinatorial Chemistry & High
Throughput Screening, 2008, 11, 357-369; Y. Yang, Non-invasive
Optical Biosensor for Probing Cell Signaling, in Sensors, 2007,
Volume 7, 2316-2329).
An RWG biosensor consists of a substrate (e.g., glass), a waveguide
thin film with an embedded grating structure, and a cell layer. The
RWG biosensor utilizes the resonant coupling of light into a
waveguide by means of a diffraction grating, leading to total
internal reflection at the solution-surface interface. This type of
biosensor can be used in cell-based assays to monitor changes in
cell morphology, cell adhesion or other cell status parameters. The
system for using such RWG biosensors can be divided into those
based on angle-shift or wavelength-shift measurements. In a
wavelength-shift measurement, polarized light covering a range of
incident wavelengths with a constant angle is used to illuminate
the waveguide; light at specific wavelengths is coupled into and
propagates along the waveguide. Alternatively, in angle-shift
instruments, the sensor is illuminated with monochromatic light and
the angle at which light is resonantly coupled is measured. The
resonance conditions are influenced by the physical properties of
the cell layer that contacts with the surface of a biosensor (e.g.,
cell confluency, adhesion and status such as proliferating or
quiescent states).
Such optical biosensor in the form of resonant waveguide,
consisting of a substrate with an optical grating and a coating
with a high refraction index, may be used as label-free means to
monitor the beating of cardiomyocytes by monitoring the change in
refractive index upon cell-substrate interaction during
cardiomyocyte contraction and relaxation cycle (i.e. beating
cycle). The method for such label-free monitoring of beating of
cardiomyocytes includes providing a RWG biosensor for monitoring
cell status operably connected to angle-shift or wavelength shift
measurement system, where the device includes at least two wells
optionally coated with fibronectin (or other extracellular matrix
proteins) to expedite attachment; adding cells to the at least two
wells, where the cells can be mouse or human or other mammalian ES
cells destined to differentiate into cardiomyocytes or primary
cardiomyocytes isolated directly from the heart of an experimental
system including mice, rats, rabbits or dog; optically monitoring
the cells of at least two wells at time intervals over a period of
time via the measurement of wavelength shift or angle-shift,
optionally calculating average rate of beats per unit time, average
amplitude intensity in a unit time as well as the average length of
time between the beats.
EXAMPLES
Extra-Cellular Recording and Cell-Substrate Impedance Measurement
of Stem-Cell Derived Cardiomyocytes
Example 1
Extra-Cellular Recording on a Disc-Shaped Microelectrode Array
Medium and Reagents
The standard cell culture medium was Cor.At.RTM. Culture Medium
(AXIOGENESIS AG, Cologne, Germany) supplemented with 5% of fetal
bovine serum (Hyclone, Logan, USA), 100 .mu.g/ml of puromycin.
Fibronectin from bovine plasma (1 mg/mL solution, Sigma, St. Louis,
USA) was used as coating (4 hrs or overnight) material before
plating the mouse embryonic stem cell. E4031, a class III
antiarrhythmic drug which is a specific antagonist for hERG and EAG
like channels was obtained from Sigma (St. Louis, USA).
Cardiomyocyte Preparation
Cardiomyocytes (Cor.At.RTM., AXIOGENESIS AG, Cologne, Germany) are
derived from transgenic mouse embryonic stem cells. Each vial
contains 1 million viable Cor.At.RTM. cardiomyocytes (>99.9%
pure). These highly purified cardiomyocyte maintain the phenotype
of adult mouse cardiomyocyte and express cardiac-specific
connexin-43, which is an indication of the ability for
excitation-contraction coupling of differentiated cardiomyocyte in
vitro.
The stem cells were thawed and the suspension drop of cell density
is around 10.sup.6-7/ml. The thawed stem cells were plated onto
fibronectin coated multi-electrodes chip which is embed in PP dish
(.about.130 ml volume) as the bottom. The cell plated chips were
placed in 37.degree. C. incubator (with 5% CO2, 95% humidity). The
cell culture medium was changed once a day. The plated Cor.At.RTM.
cell line can be grown up to 21 days post thaw with medium changes
every other day.
Multichannel Recording
After three to four days growing in 37.degree. C. incubator (with
5% CO2, 95% humidity), the differentiated cells formed a monolayer
onto the multi-electrode chip with some congregated cardiomyocyte
showing rhythmically self-beating. The earliest onset of self
beating was found on the second day after cell plating. The
multichannel extracellular recording was carried out on day 3 to
day 5 after cell plating using WPI ISO-DAM8A (eight channel module)
amplifier (World Precision Instruments, Sarasota, USA) and Dataq
DI-0720 data acquisition interface (Dataq Instrument Inc., Akron,
USA). The multi-electrode chip contains an array of 30 electrodes
(FIG. 17), and the diameter of the electrode tip is 50 .mu.m and
the interval between each two neighbor electrode tips is 50 .mu.m.
The signals were collected via the 4 electrodes in center which
were arranged in a rectangle shape (A2, B2, C2 and D2 as shown in
FIG. 17). For extracellular field potential, the field potential is
measured between each of the 4-electrodes and an electrical
connection that connects to all the other 26 electrodes. The
extracellular field potential (FP) data were collected with
following parameters being used: Low cut filter: 5 Hz; High cut
filter: 1 K Hz; Sampling rate: 5 K Hz; Display sensitivity: 10
K.
Results
General Features of Field Potential
The FP signal is relatively small; the peak to peak amplitude is
.ltoreq.1 mV. During the early process (day 2 to day 4 after cell
plating) of growing and differentiating, the self beating frequency
of cardiomyocyte cluster was increased from 60 to 90/min on day 2
(n=4) to 160 to 220/min on day 4 (n=4), and the beating frequency
remain nearly-constant after day 4. In the meantime, the duration
of FP was reduced and the depolarizing velocity which reflecting
the upstroke of the FP was shortened while the amplitude of the FP
was increased (FIG. 18). As shown in FIG. 18, the
electrophysiological features were changed as the mouse derived
stem cell grows and develops on day 2 and day 3. The FP amplitude
and frequency were increased while the duration and depolarizing
velocity were shortened.
Since the extracellular FP is the negative reflection of the
intracellular voltage fluctuation, the standard shape of FP
waveform consists downward depolarization peak followed by upward
repolarization peak. In actual recording, we observed an initial
upward peak and downward peak followed by an upward peak (FIG.
18).
Effect of E4031 on FP
E4031, antiarrhythmic agent, is a specific antagonist for hERG and
EAG channel family which participate repolarization during action
potential phase 3. So In present study, we picked FP duration as
our main target index, and it directly correlated to the "QT
interval" in clinical ECG. We found E4031 concentration-dependently
delayed the depolarizing velocity and prolonged FP duration in
mouse cardiomyocyte (FIG. 19) with IC.sub.50 of 405 nM (FIG. 20).
The on-site action of E 4031 on field potential is a slightly slow
and it possibly due relatively the low binding affinity of E4031 to
its receptor. At a concentration of 75 to 150 nM, E4031 exerted
"anti-arrhythmic" action on field potential frequency in the
mouse-stem-cell derived cardiomyocytes (FIG. 21).
Example 2
Extra-Cellular Recording Using One Electrode Structure on the
Substrate and One External Reference Electrode
FIG. 22 shows an extra-cellular field potential recording of mouse
stem-cell derived cardiomyocytes obtained using a device of the
present invention. The circle-on-line electrode array (with circle
diameter 90 micron, line width 30 micron, the electrode gap
distance 20 micron, see FIG. 13F for representation of
circle-on-line electrodes) consisting of two electrode structures
are fabricated on a glass substrate. During extra-cellular field
potential recording, one circle-on-line electrode is used as a
recording electrode and an external wire electrode is inserted into
the well serving as a reference electrode. Cell preparation,
reagents, cell culture and extracellular recording setup are the
same as those described in to example 1.
FIG. 22A shows a field potential (after an amplification of 10,000)
before treatment for a 3-day cardiomyocyte culture in a well
containing circle-on-line electrodes.
Cardiomyocytes were beating at a rate of .about.91 beats per
minute. At the middle point of the FIG. 22A, E4031 was added to the
well. No immediate effect was observed after the treatment with
E4031. However, with time, the field potential gradually became
irregular. The irregular field potentials at .about.18 seconds and
.about.3 minutes after treatment were shown on FIGS. 22B and 22C.
At .about.5.5 minutes after treatment, the field potential
magnitude was significantly reduced and later on, no field
potential peaks could be detected.
Example 3
Parallel Cell-Impedance Measurement and Extra-Cellular Recording
for Cardiomyocytes Prior to and after Qunindine Treatment
FIG. 23A through 23E show another example of field potential change
for cardiomyocytes at different time points before and after the
treatment with 3 uM Quinidine, as recorded from a 3-day
cardiomyocyte culture in a well containing circle-on-electrodes.
Similar to the configuration for data in FIG. 22, an external wire
electrode was inserted to the well serving as a reference
electrode. Before treatment, cardiomyocytes were beating at a rate
of .about.72 beats per minute. As shown in FIG. 23 B through D,
corresponding to the field potential recorded at 10 seconds, 50
seconds, 3 minutes and 9 minutes after treatment with 3 uM
Quindine, Quindine has a dramatic effect on the field potential of
the cardiomyocytes.
In parallel, impedance of the cardiomyocytes in such a well is
monitored. FIG. 24A shows the impedance spikes as monitored on the
circle-on-line electrode array before treatment. As shown in FIG.
24B through D, corresponding to the impedance spike pattern
measured at .about.1.5 minute, .about.3 minute and .about.11 minute
after treatment with 3 uM Quinidine, impedance-based monitoring can
readily detect the change in cardiomyocyte beating function as a
result of Quindine treatment. Notably, at .about.11 minutes after
Quindine treatment, the amplitude of impedance spikes significantly
reduced and the impedance spiking also becomes very irregular.
Example 4
Extra-Cellular Recording for Primary Cardiomyocytes Between a
Circle-on-Line Electrode Structure and an Externally Applied
Reference Electrode
Primary cardiomyocytes were prepared from by perfusing sacrificed
mice' heart. The primary cardiomyocytes were added to a well
containing a circle-on-line electrode array (with circle diameter
90 micron, line width 30 micron, the electrode gap distance 20
micron) on the well bottom. The electrode array is fabricated on a
glass substrate. Before seeding the cells, the well was pre-coated
with fibronectin. Each well was seeded with about 40,000 cells and
was measured between 48 and 72 hrs after seeding. During
extra-cellular field potential recording, one circle-on-line
electrode structure is used as a recording electrode and an
external wire electrode is inserted into the well serving as a
reference electrode. The setting for extra-cellular recording is
the same as those used in Example 1. FIG. 25 shows a typical field
potential from such a primary cardiomyocyte culture.
Example 5
Parallel Cell-Impedance Measurement and Extra-Cellular Recording
for Cardiomyocytes
FIG. 27 shows the design of an electrode array used for combined
cell-impedance measurement and extra-cellular recording. For this
design, the electrode array consists of two extracellular-recording
electrodes 2701 and 2702 with circle geometry having diameters of
60 microns, located on either left or right side of, and in the
middle section the array. Note that the recording electrode
diameter could be varied from being as small as 5 micron to as
large as 0.5 mm, even 1 mm, or even multiple millimeters. Electrode
array further consists of two electrode structures 2703 and 2704,
each comprising multiple electrode elements. Electrode elements
within each electrode structure are of circle-on-line geometry and
comprise interconnected circles having a diameter of 90 microns
positioned along a straight line of 30 micron width (the electrode
gap distance 20 micron, see FIG. 13F for representation of
circle-on-line electrodes). Electrode element 2705 is the first
element counting from top for electrode structure 2703. Electrode
element 2706 is the first element counting from top for electrode
structure 2704. Electrode elements on electrode structure 2703 are
connected together through electrode bus 2707, having a width of
0.2 mm, to a connection pad 2709 which is located on the left side
of FIG. 27. Similarly, electrode elements of the electrode
structure 2704 are connected together through electrode bus 2708,
having a width of 0.2 mm, to a connection pad 2710 which is located
on the right side of FIG. 27. The overall electrode area excluding
the regions consisting of electrode buses 2707 and 2708 has a
diameter of 3.1 mm.
The electrode array of FIG. 27 is fabricated on glass substrate
using photolithography methods for pattern-generation of thin gold
electrode arrays (gold film thickness of .about.100 nm on top of a
Cr adhesion layer of .about.10 nm). A bottomless well plate having
a well-diameter of 5 mm is assembled to the electrodes-containing
glass slides, with the electrode array shown on FIG. 27 being
positioned at the central region of the well.
For cell impedance measurement, the impedance between two electrode
structures 2703 and 2704 is measured by connecting an ACEA
real-time-cell-analysis impedance analyzer capable of millisecond
time resolution (e.g., 15 milliseconds or less impedance data
update rate for measuring 96 wells simultaneously) to the two
connection pads 2709 and 2710, connected to responding electrode
buses 2707 and 2708, respectively. For extracellular recording
(ECR), various recording modes can be used. (1) ECR mode 1 is used
to for monitoring electrode electrical voltage signals between an
ECR electrode 2701 (or 2702) and one (impedance-measurement)
electrode structure 2703 (or 2704) by connecting an ECR connection
pad (2711 or 2712) and an impedance-measurement electrode
connection pad (2709 or 2710) to a voltage amplifier. For this
mode, extracellular signals from ECR electrodes are measured with
the impedance-measurement electrode structure 2703 or 2704 used as
"reference electrode". The impedance-measurement electrode
structure 2703 (or 2704) has a much smaller electrode-impedance due
to its much larger surface area (over 100 times larger) than ECR
electrodes 2701 (or 2702). (2) ECR mode 2 is used for monitoring
electrode electrical voltage signals between an ECR electrode 2701
(or 2702) and an externally applied gold reference electrode (not
shown on FIG. 27) inserted into the well by connecting an ECR
connection pad (2711 or 2712) and the gold external electrode to a
voltage amplifier. For this mode, extracellular signals from ECR
electrodes are measured with the external gold electrode used as
"reference electrode". Such external gold electrode will not have
any cardiomyocytes attached and would serve as cell-free reference
electrodes. (3) ECR mode 3 is used for monitoring electrical
voltage signals between impedance-measurement electrode structure
2703 (or 2704) and an externally applied gold reference electrode
(not shown on FIG. 27) inserted into the well by connecting an
impedance-electrode connection pad (2709 or 2710) and the external
gold electrode to a voltage amplifier. For this mode, extracellular
signals from impedance-measurement electrodes are measured with the
external gold electrode used as "reference electrode". Such
external gold electrode will not have any cardiomyocytes attached
and would serve as cell-free reference electrodes.
FIGS. 28 and 29 are examples of impedance measurement and
extracellular recording of Cor. At cardiomyocytes. Cor. At cells
were prepared and cultured using the same method as those described
in Example 1. The data on FIGS. 28 and 29 is for the Cor. At cells
on day 5 after cell seeding into the electrodes-containing wells.
FIG. 28 shows the impedance beating result obtained for the Cor. At
cells for .about.12 seconds with time-solution about 28.8 ms per
data point. The impedance data is represented with cell index, a
dimension less parameter. FIG. 29 shows the ECR voltage signals for
the three different recording modes described above, including ECR
electrode vs impedance electrode-structures, ECR electrode vs
external gold electrode and impedance electrode-structure vs
external gold electrode. The voltage amplifier and other ECR
details are the same as those discussed in Example 1. In brief, the
multichannel extracellular recording was carried out using WPI
ISO-DAM8A (eight channel module) amplifier (World Precision
Instruments, Sarasota, USA) and Dataq DI-0720 data acquisition
interface (Dataq Instrument Inc., Akron, USA). The extracellular
field potential data were collected with following parameters being
used: voltage signal gain: 10,000; Low cut filter: 1 Hz; High cut
filter: 1 K Hz; Sampling rate: 2-5 K Hz.
Example 6
Parallel Cell-Impedance Measurement and Extra-Cellular Recording
for Cardiomyocytes
FIG. 30 shows the design of another electrode array used for
combined cell-impedance measurement and extra-cellular recording.
For this design, the electrode array consists of two
extracellular-recording electrodes 3001 and 3002 with circle
geometry having diameters of 60 microns, located in the middle
section the array. Note that the recording electrode diameter could
be varied from being as small as 5 micron to as large as 0.5 mm,
even 1 mm, or even multiple millimeters. ECR electrodes 3001 and
3002 are connected to the connection pads 3011 and 3012,
respectively. The electrode array further consists of two electrode
structures 3003 and 3004, each comprising multiple electrode
elements. Electrode elements within each electrode structure are of
circle-on-arc (curved line) geometry and comprise interconnected
circles having a diameter of 90 microns positioned along curve
lines of 30 micron width (the electrode gap distance 20 micron, see
FIG. 13F for representation of circle-on-line electrodes). The
first electrode element of the electrode structure 3003, counting
from top of the electrode array, has many circles
aligned-on-curved-line, forming a semi-circle arc. The first
electrode element of the electrode structure 3004, counting from
bottom of the electrode array, has many circles aligned-on-curved
line, forming a semi-circle arc. Electrode elements on electrode
structure 3005 are connected together through horizontal lines
located in the middle of the electrode array to the connection pad
3009 which is located on the left side of the electrode array.
Electrode elements on electrode structure 3006 are connected
together through horizontal lines located in the middle of the
electrode array to the connection pad 3010 which is located on the
right side of the electrode array.
The electrode array of FIG. 28 is fabricated on glass substrate
using photolithography methods for pattern-generation of thin gold
electrode arrays (gold film thickness of 100 nm on top of a Cr
adhesion layer of 10 nm). A bottomless well plate having a
well-diameter of 5 mm is assembled to the electrodes-containing
glass slides, with the electrode array shown on FIG. 30 being
positioned at the central region of the well.
For cell impedance measurement, the impedance between two electrode
structures 3003 and 3004 is measured by connecting an ACEA
real-time-cell-analysis impedance analyzer capable of millisecond
time resolution (e.g., 15 milliseconds or less impedance data
update rate for measuring 96 wells simultaneously) to the two
connection pads 3009 and 3010, connected to responding electrode
structures 3003 and 3004, respectively. For extracellular recording
(ECR), various recording modes can be used. (1) ECR mode 1 is used
to for monitoring electrode electrical voltage signals between an
ECR electrode 3001 (or 3002) and one (impedance-measurement)
electrode structure 3003 (or 3004) by connecting an ECR connection
pad (3011 or 3012) and an impedance-measurement electrode
connection pad (3009 or 3010) to a voltage amplifier. For this
mode, extracellular signals from ECR electrodes are measured with
the impedance-measurement electrode structure 3003 or 3004 used as
"reference electrode". The impedance-measurement electrode
structure 3003 (or 3004) has a much smaller electrode-impedance due
to its much larger surface area (over 100 times larger) than ECR
electrodes 3001 (or 3002). (2) ECR mode 2 is used for monitoring
electrode electrical voltage signals between an ECR electrode 3001
(or 3002) and an externally applied gold reference electrode (not
shown on FIG. 30) inserted into the well by connecting an ECR
connection pad (3011 or 3012) and the gold external electrode to a
voltage amplifier. For this mode, extracellular signals from ECR
electrodes are measured with the external gold electrode used as
"reference electrode". Such external gold electrode will not have
any cardiomyocytes attached and would serve as cell-free reference
electrodes. (3) ECR mode 3 is used for monitoring electrical
voltage signals between impedance-measurement electrode structure
3003 (or 3004) and an externally applied gold reference electrode
(not shown on FIG. 30) inserted into the well by connecting an
impedance-electrode connection pad (3009 or 3010) and the external
gold electrode to a voltage amplifier. For this mode, extracellular
signals from impedance-measurement electrodes are measured with the
external gold electrode used as "reference electrode". Such
external gold electrode will not have any cardiomyocytes attached
and would serve as cell-free reference electrodes.
FIGS. 31 and 32 are examples of impedance measurement and
extracellular recording of Cor. At cardiomyocytes. Cor. At cells
were prepared and cultured using the same method as those described
in Example 1. The data on FIGS. 31 and 32 is for the Cor. At cells
on day 5 after cell seeding into the electrodes-containing wells.
FIG. 31 shows the impedance beating result obtained for the Cor. At
cells for .about.12 seconds with time-solution about 28.8 ms per
data point. The impedance data is represented with cell index, a
dimension less parameter. FIG. 32 shows the ECR voltage signals for
the three different recording modes described above, including ECR
electrode vs impedance electrode-structures, ECR electrode vs
external gold electrode and impedance electrode-structure vs
external gold electrode. The voltage amplifier and other ECR
details are the same as those discussed in Example 1. In brief, the
multichannel extracellular recording was carried out using WPI
ISO-DAM8A (eight channel module) amplifier (World Precision
Instruments, Sarasota, USA) and Dataq DI-0720 data acquisition
interface (Dataq Instrument Inc., Akron, USA). The extracellular
field potential data were collected with following parameters being
used: voltage signal gain: 10,000; Low cut filter: 1 Hz; High cut
filter: 1 K Hz; Sampling rate: 2-5 K Hz.
* * * * *
References